Modified glucosidase, beta, acid polynucleotides for treating protein deficiency

ABSTRACT

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/791,922, filed Mar. 9, 2013, entitled Modified Polynucleotides forthe Production of Biologics and Proteins Associated with Human Diseasewhich claims priority to U.S. Provisional Patent Application No.61/681,742, filed, Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Oncology-Related Proteins and Peptides, U.S.Provisional Patent Application No. 61/737,224, filed Dec. 14, 2012,entitled Terminally Optimized Modified RNAs, International ApplicationNo PCT/US2012/069610, filed Dec. 14, 2012, entitled Modified Nucleoside,Nucleotide, and Nucleic Acid Compositions, U.S. Provisional PatentApplication No. 61/618,862, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Biologics, U.S. Provisional PatentApplication No. 61/681,645, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Biologics, U.S. Provisional PatentApplication No. 61/737,130, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Biologics, U.S. Provisional PatentApplication No. 61/618,866, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Antibodies, U.S. ProvisionalPatent Application No. 61/681,647, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Antibodies, U.S.Provisional Patent Application No. 61/737,134, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Antibodies, U.S.Provisional Patent Application No. 61/618,868, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Vaccines, U.S.Provisional Patent Application No. 61/681,648, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Vaccines, U.S.Provisional Patent Application No. 61/737,135, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Vaccines, U.S.Provisional Patent Application No. 61/618,870, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of TherapeuticProteins and Peptides, U.S. Provisional Patent Application No.61/681,649, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Therapeutic Proteins and Peptides, U.S. ProvisionalPatent Application No. 61/737,139, filed Dec. 14, 2012, ModifiedPolynucleotides for the Production of Therapeutic Proteins and Peptides,U.S. Provisional Patent Application No. 61/618,873, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of SecretedProteins, U.S. Provisional Patent Application No. 61/681,650, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofSecreted Proteins, U.S. Provisional Patent Application No. 61/737,147,filed Dec. 14, 2012, entitled Modified Polynucleotides for theProduction of Secreted Proteins, U.S. Provisional Patent Application No.61/618,878, filed Apr. 2, 2012, entitled Modified Polynucleotides forthe Production of Plasma Membrane Proteins, U.S. Provisional PatentApplication No. 61/681,654, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Plasma Membrane Proteins, U.S.Provisional Patent Application No. 61/737,152, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins, U.S. Provisional Patent Application No. 61/618,885, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/681,658, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins, U.S. Provisional Patent Application No. 61/737,155, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/618,896, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins, U.S. Provisional Patent Application No. 61/668,157, filed Jul.5, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/681,661, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins, U.S. Provisional Patent Application No. 61/737,160, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/618,911, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Nuclear Proteins, U.S. ProvisionalPatent Application No. 61/681,667, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Nuclear Proteins, U.S.Provisional Patent Application No. 61/737,168, field Dec. 14, 2012,entitled Modified Polynucleotides for the Production of NuclearProteins, U.S. Provisional Patent Application No. 61/618,922, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/681,675, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/737,174, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Patent Application No. 61/618,935, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/681,687, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/737,184, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/618,945, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/681,696, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/737,191, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/618,953, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/681,704, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/737,203, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/618,961, filed Apr. 2, 2012, entitled Dosing Methodsfor Modified mRNA, U.S. Provisional Patent Application No. 61/648,286,filed May 17, 2012, entitled Dosing Methods for Modified mRNA, thecontents of each of which are herein incorporated by reference in itsentirety.

This application is also related to International Publication No.PCT/US2012/58519, filed Oct. 3, 2012, entitled Modified Nucleosides,Nucleotides, and Nucleic Acids, and Uses Thereof and InternationalPublication No. PCT/US2012/69610, filed Dec. 14, 2012, entitled ModifiedNucleoside, Nucleotide, and Nucleic Acid Compositions.

The instant application is also related to co-pending applications, eachfiled concurrently herewith on Mar. 9, 2013 and having Attorney DocketNumber M301.20, (PCT/US13/030,063) entitled Modified Polynucleotides;Attorney Docket Number M304.20 (PCT/US13/030,064), entitled ModifiedPolynucleotides for the Production of Secreted Proteins; Attorney DocketNumber M305.20 (PCT/US13/030,059), entitled Modified Polynucleotides forthe Production of Membrane Proteins; Attorney Docket Number M306.20(PCT/US13/030,066), entitled Modified Polynucleotides for the Productionof Cytoplasmic and Cytoskeletal Proteins; Attorney Docket Number M308.20(PCT/US13/030,067), entitled Modified Polynucleotides for the Productionof Nuclear Proteins; Attorney Docket Number M309.20 (PCT/US13/030,060),entitled Modified Polynucleotides for the Production of Proteins;Attorney Docket Number M310.20 (PCT/US13/030,061), entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease; Attorney Docket Number MNC1.20 (PCT/US13/030,068), entitledModified Polynucleotides for the Production of Cosmetic Proteins andPeptides and Attorney Docket Number MNC2.20 (PCT/US13/030,070), entitledModified Polynucleotides for the Production of Oncology-Related Proteinsand Peptides, the contents of each of which are herein incorporated byreference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing file entitledM300USCON11SQLST.txt, was created on Feb. 3, 2014 and is 48,972,678bytes in size. The information in electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to compositions, methods, processes, kits anddevices for the design, preparation, manufacture and/or formulation ofpolynucleotides, primary constructs and modified mRNA molecules (mmRNA).

BACKGROUND OF THE INVENTION

There are multiple problems with prior methodologies of effectingprotein expression. For example, introduced DNA can integrate into hostcell genomic DNA at some frequency, resulting in alterations and/ordamage to the host cell genomic DNA. Alternatively, the heterologousdeoxyribonucleic acid (DNA) introduced into a cell can be inherited bydaughter cells (whether or not the heterologous DNA has integrated intothe chromosome) or by offspring. In addition, assuming proper deliveryand no damage or integration into the host genome, there are multiplesteps which must occur before the encoded protein is made. Once insidethe cell, DNA must be transported into the nucleus where it istranscribed into RNA. The RNA transcribed from DNA must then enter thecytoplasm where it is translated into protein. Not only do the multipleprocessing steps from administered DNA to protein create lag timesbefore the generation of the functional protein, each step represents anopportunity for error and damage to the cell. Further, it is known to bedifficult to obtain DNA expression in cells as DNA frequently enters acell but is not expressed or not expressed at reasonable rates orconcentrations. This can be a particular problem when DNA is introducedinto primary cells or modified cell lines.

In the early 1990's Bloom and colleagues successfully rescuedvasopressin-deficient rats by injecting in vitro-transcribed vasopressinmRNA into the hypothalamus (Science 255: 996-998; 1992). However, thelow levels of translation and the immunogenicity of the moleculeshampered the development of mRNA as a therapeutic and efforts have sincefocused on alternative applications that could instead exploit thesepitfalls, i.e. immunization with mRNAs coding for cancer antigens.

Others have investigated the use of mRNA to deliver a polypeptide ofinterest and shown that certain chemical modifications of mRNAmolecules, particularly pseudouridine and 5-methyl-cytosine, havereduced immunostimulatory effect.

These studies are disclosed in, for example, Ribostem Limited in UnitedKingdom patent application serial number 0316089.2 filed on Jul. 9, 2003now abandoned, PCT application number PCT/GB2004/002981 filed on Jul. 9,2004 published as WO2005005622, United States patent applicationnational phase entry Ser. No. 10/563,897 filed on Jun. 8, 2006 publishedas US20060247195 now abandoned, and European patent application nationalphase entry serial number EP2004743322 filed on Jul. 9, 2004 publishedas EP1646714 now withdrawn; Novozymes, Inc. in PCT application numberPCT/US2007/88060 filed on Dec. 19, 2007 published as WO2008140615,United States patent application national phase entry Ser. No.12/520,072 filed on Jul. 2, 2009 published as US20100028943 and Europeanpatent application national phase entry serial number EP2007874376 filedon Jul. 7, 2009 published as EP2104739; University of Rochester in PCTapplication number PCT/US2006/46120 filed on Dec. 4, 2006 published asWO2007064952 and U.S. patent application Ser. No. 11/606,995 filed onDec. 1, 2006 published as US20070141030; BioNTech AG in European patentapplication serial number EP2007024312 filed Dec. 14, 2007 nowabandoned, PCT application number PCT/EP2008/01059 filed on Dec. 12,2008 published as WO2009077134, European patent application nationalphase entry serial number EP2008861423 filed on Jun. 2, 2010 publishedas EP2240572, United States patent application national phase entry Ser.No. 12/,735,060 filed Nov. 24, 2010 published as US20110065103, Germanpatent application serial number DE 10 2005 046 490 filed Sep. 28, 2005,PCT application PCT/EP2006/0448 filed Sep. 28, 2006 published asWO2007036366, national phase European patent EP1934345 published March,21, 2012 and national phase U.S. patent application Ser. No. 11/992,638filed Aug. 14, 2009 published as 20100129877; Immune Disease InstituteInc. in U.S. patent application Ser. No. 13/088,009 filed Apr. 15, 2011published as US20120046346 and PCT application PCT/US2011/32679 filedApr. 15, 2011 published as WO20110130624; Shire Human GeneticTherapeutics in U.S. patent application Ser. No. 12/957,340 filed onNov. 20, 2010 published as US20110244026; Sequitur Inc. in PCTapplication PCT/US1998/019492 filed on Sep. 18, 1998 published asWO1999014346; The Scripps Research Institute in PCT application numberPCT/US2010/00567 filed on Feb. 24, 2010 published as WO2010098861, andUnited States patent application national phase entry Ser. No.13/203,229 filed Nov. 3, 2011 published as US20120053333;Ludwig-Maximillians University in PCT application numberPCT/EP2010/004681 filed on Jul. 30, 2010 published as WO2011012316;Cellscript Inc. in U.S. Pat. No. 8,039,214 filed June 30, 2008 andgranted Oct. 18, 2011, U.S. patent application Ser. No. 12/962,498 filedon Dec. 7, 2010 published as US20110143436, Ser. No. 12/962,468 filed onDec. 7, 2010 published as US20110143397, Ser. No. 13/237,451 filed onSep. 20, 2011 published as US20120009649, and PCT applicationsPCT/US2010/59305 filed Dec. 7, 2010 published as WO2011071931 andPCT/US2010/59317 filed on Dec. 7, 2010 published as WO2011071936; TheTrustees of the University of Pennsylvania in PCT application numberPCT/US2006/32372 filed on Aug. 21, 2006 published as WO2007024708, andUnited States patent application national phase entry Ser. No.11/990,646 filed on Mar. 27, 2009 published as US20090286852; CurevacGMBH in German patent application serial numbers DE10 2001 027 283.9filed Jun. 5, 2001, DE10 2001 062 480.8 filed Dec. 19, 2001, and DE 202006 051 516 filed Oct. 31, 2006 all abandoned, European patent numbersEP1392341 granted Mar. 30, 2005 and EP1458410 granted Jan. 2, 2008, PCTapplication numbers PCT/EP2002/06180 filed Jun. 5, 2002 published asWO2002098443, PCT/EP2002/14577 filed on Dec. 19, 2002 published asWO2003051401, PCT/EP2007/09469 filed on Dec. 31, 2007 published asWO2008052770, PCT/EP2008/03033 filed on Apr. 16, 2008 published asWO2009127230, PCT/EP2006/004784 filed on May 19, 2005 published asWO2006122828, PCT/EP2008/00081 filed on Jan. 9, 2007 published asWO2008083949, and U.S. patent application Ser. No. 10/729,830 filed onDec. 5, 2003 published as US20050032730, Ser. No. 10/870,110 filed onJun. 18, 2004 published as US20050059624, Ser. No. 11/914,945 filed onJul. 7, 2008 published as US20080267873, Ser. No. 12/446,912 filed onOct. 27, 2009 published as US2010047261 now abandoned, 12/522,214 filedon Jan. 4, 2010 published as US20100189729, Ser. No. 12/787,566 filed onMay 26, 2010 published as US20110077287, Ser. No. 12/787,755 filed onMay 26, 2010 published as US20100239608, Ser. No. 13/185,119 filed onJul. 18, 2011 published as US20110269950, and Ser. No. 13/106,548 filedon May 12, 2011 published as US20110311472 all of which are hereinincorporated by reference in their entirety.

Notwithstanding these reports which are limited to a selection ofchemical modifications including pseudouridine and 5-methyl-cytosine,there remains a need in the art for therapeutic modalities to addressthe myriad of barriers surrounding the efficacious modulation ofintracellular translation and processing of nucleic acids encodingpolypeptides or fragments thereof.

To this end, the inventors have shown that certain modified mRNAsequences have the potential as therapeutics with benefits beyond justevading, avoiding or diminishing the immune response. Such studies aredetailed in published co-pending applications International ApplicationPCT/US2011/046861 filed Aug. 5, 2011 and PCT/US2011/054636 filed Oct. 3,2011, International Application number PCT/US2011/054617 filed Oct. 3,2011, the contents of which are incorporated herein by reference intheir entirety.

The present invention addresses this need by providing nucleic acidbased compounds or polynucleotides which encode a polypeptide ofinterest (e.g., modified mRNA or mmRNA) and which have structural and/orchemical features that avoid one or more of the problems in the art, forexample, features which are useful for optimizing formulation anddelivery of nucleic acid-based therapeutics while retaining structuraland functional integrity, overcoming the threshold of expression,improving expression rates, half life and/or protein concentrations,optimizing protein localization, and avoiding deleterious bio-responsessuch as the immune response and/or degradation pathways.

SUMMARY OF THE INVENTION

Described herein are compositions, methods, processes, kits and devicesfor the design, preparation, manufacture and/or formulation of modifiedmRNA (mmRNA) molecules.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 is a schematic of a primary construct of the present invention.

FIG. 2 illustrates lipid structures in the prior art useful in thepresent invention. Shown are the structures for 98N12-5 (TETA5-LAP),DLin-DMA, DLin-K-DMA(2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA,DLin-MC3-DMA and C12-200.

FIG. 3 is a representative plasmid useful in the IVT reactions taughtherein. The plasmid contains Insert 64818, designed by the instantinventors.

FIG. 4 is a gel profile of modified mRNA encapsulated in PLGAmicrospheres.

FIG. 5 is a histogram of Factor IX protein production PLGA formulationFactor IX modified mRNA.

FIG. 6 is a histogram showing VEGF protein production in humankeratinocyte cells after transfection of modified mRNA at a range ofdoses. FIG. 6A shows protein production after transfection of modifiedmRNA comprising natural nucleoside triphosphate (NTP). FIG. 6B showsprotein production after transfection of modified mRNA fully modifiedwith pseudouridine (Pseudo-U) and 5-methylcytosine (5mC). FIG. 6C showsprotein production after transfection of modified mRNA fully modifiedwith N1-methyl-pseudouridine (N1-methyl-Pseudo-U) and 5-methylcytosine(5mC).

FIG. 7 is a histogram of VEGF protein production in HEK293 cells.

FIG. 8 is a histogram of VEGF expression and IFN-alpha induction aftertransfection of VEGF modified mRNA in peripheral blood mononuclear cells(PBMC).

FIG. 8A shows VEGF expression. FIG. 8B shows IFN-alpha induction.

FIG. 9 is a histogram of VEGF protein production in HeLa cells from VEGFmodified mRNA.

FIG. 10 is a histogram of VEGF protein production from lipoplexed VEGFmodified mRNA in mice.

FIG. 11 is a histogram of G-CSF protein production in HeLa cells fromG-CSF modified mRNA.

FIG. 12 is a histogram of G-CSF protein production in mice fromlipoplexedG-CSF modified mRNA.

FIG. 13 is a histogram of Factor IX protein production in HeLa cellsupernatant from Factor IX modified mRNA.

FIG. 14 is a histogram of APOA1 protein production in HeLa cells fromAPOA1 wild-type modified mRNA, APOA1 Milano modified mRNA or APOA1 Parismodified mRNA.

FIG. 15 is a gel profile of APOA1 protein from APOA1 wild-type modifiedmRNA.

FIG. 16 is a gel profile of APOA1 protein from APOA1 Paris modifiedmRNA.

FIG. 17 is a gel profile of APOA1 protein from APOA1 Milano modifiedmRNA.

FIG. 18 is a gel profile of Fibrinogen alpha (FGA) protein from FGAmodified mRNA.

FIG. 19 is a histogram of Plasminogen protein production in HeLa cellsupernatant from Plasminogen modified mRNA.

FIG. 20 is a gel profile of Plasminogen protein from Plasminogenmodified mRNA.

FIG. 21 is a gel profile of galactose-1-phosphate uridylyltransferase(GALT) protein from GALT modified mRNA.

FIG. 22 is a gel profile of argininosuccinate lyase (ASL) protein fromASL modified mRNA.

FIG. 23 is a gel profile of tyrosine aminotransferase (TAT) protein fromTAT modified mRNA.

FIG. 24 is a gel profile of glucan (1,4-alpha-), branching enzyme 1(GBE1) protein from GBE1 modified mRNA.

FIG. 25 is a histogram of Prothrombin protein production in HeLa cellsupernatant from Prothrombin modified mRNA.

FIG. 26 is a histogram of Prothrombin protein production in HeLa cellsupernatant from Prothrombin modified mRNA.

FIG. 27 is a gel profile of ceruloplasmin (CP or CLP) protein from CPmodified mRNA.

FIG. 28 is a histogram of transforming growth factor beta 1 (TGF-beta1)protein production in HeLa cell supernatant from TGF-beta1 modifiedmRNA.

FIG. 29 is a gel profile of ornithine carbamoyltransferase (OTC) proteinfrom OTC modified mRNA.

FIG. 30 is a flow cytometry plot of low density lipoprotein receptor(LDLR) modified mRNA.

FIG. 31 is a gel profile of UDP glucuronosyltransferase 1 family,polypeptide A1 (UGT1A1) protein from UGT1A1 modified mRNA.

FIG. 32 is a histogram of Factor XI protein production in HEK293 cells.

FIG. 33 is a gel profile of Aquaporin-5 protein from Aquaporin-5modified mRNA.

FIG. 34 is a histogram of Factor VII protein production in HeLa cellsfrom Factor VII modified mRNA.

FIG. 35 is a histogram of Insulin Glargine protein production in HeLacells from Insulin Glargine modified mRNA.

FIG. 36 is a histogram of Tissue Factor protein production in HeLa cellsfrom Tissue Factor modified mRNA.

FIG. 37 is a histogram of Factor XI protein production in HeLa cellsfrom Factor XI modified mRNA.

FIG. 38 is a histogram of Factor XI protein production in HeLa cellsupernatant from Factor XI modified mRNA.

FIG. 39 is a histogram of Insulin Aspart protein production in HeLacells from Insulin Aspart modified mRNA.

FIG. 40 is a histogram of Insulin Lispro protein production in HeLacells from Insulin Lispro modified mRNA.

FIG. 41 is a histogram of Insulin Glulisine protein production in HeLacells from Insulin Glulisine modified mRNA.

FIG. 42 is a histogram of human growth hormone protein production inHeLa cells from human growth hormone modified mRNA.

FIG. 43 is a gel profile of tumor protein 53 (p53) protein from p53modified mRNA. FIG. 43A shows the expected size of p53. FIG. 43B showsthe expected size of p53.

FIG. 44 is a gel profile of tuftelin (TUFT1) protein from TUFT1 modifiedmRNA.

FIG. 45 is a gel profile of galactokinase 1 (GALK1) protein from GALK1modified mRNA. FIG. 45A shows the expected size of GALK1. FIG. 45B showsthe expected size of GALK1.

FIG. 46 is a gel profile of defensin, beta 103A (DEFB103A) protein fromDEFB103A modified mRNA.

FIG. 47 is a flow cytometry plot of LDLR modified mRNA.

FIG. 48 is a histogram of vascular endothelial growth factor expressionin HeLa.

FIG. 49 is a histogram of the cell vitality of HeLa cells transfectedwith vascular endothelial growth factor mRNA.

FIG. 50 is a histogram of Insulin Aspart protein expression.

FIG. 51 is a histogram of Insulin Glargine protein expression.

FIG. 52 is a histogram of Insulin Glulisine protein expression.

FIG. 53 is a histogram of Interleukin 7 (IL-7) protein expression.

FIG. 54 is a histogram of Erythropoietin (EPO) protein expression.

FIG. 55 is a gel profile of Lysosomal Acid Lipase protein from LysosomalAcid Lipase modified mRNA.

FIG. 56 is a gel profile of Glucocerebrosidase protein fromGlucocerebrosidase modified mRNA.

FIG. 57 is a gel profile of Iduronate 2-Sulfatase protein from Iduronate2-Sulfatase modified mRNA.

FIG. 58 is a gel profile of Luciferase protein from Luciferase modifiedmRNA.

FIG. 59 is a histogram of IgG concentration after administration withformulated Herceptin modified mRNA in mammals.

FIG. 60 is a histogram of IgG concentration (in ng/ml) aftertransfection with Herceptin modified mRNA.

FIG. 61 is a gel profile of Herceptin protein from Herceptin modifiedmRNA.

FIG. 62 is a histogram of Glucocerebrosidase enzyme activity.

FIG. 63 is a histogram of Lysosomal Acid Lipase enzyme activity.

FIG. 64 a is histogram of Factor VIII protein expression.

FIG. 65 is a histogram of Factor VIII Chromogenic Activity.

FIG. 66 is a graph of LDLR Expression. FIG. 66A shows LDL ReceptorExpression of cells compared to LDLR mRNA added. FIG. 66B shows LDLReceptor Expression of cells post transfection. FIG. 66C shows thesaturation of BODIPY® labeled LRL. FIG. 66D shows the binding affinityof BODIPY-LDL to cells.

FIG. 67 is a graph showing the percent positive cells for UGT1A1Expression.

FIG. 68 is a graph showing UGT1A1 protein accumulation.

FIG. 69 is a gel profile of UGT1A1 protein and OTC from eith UGT1A1 orOTC modified mRNA.

FIG. 70 is a flow cytometry plot of HEK293 cells transfected with PAh orUGT1A1.

FIG. 71 is a gel profile of UGT1A1 protein from UGT1A1 modified mRNA.

FIG. 72 is a gel profile of microsomal extracts of mice treated withLNPs containing UGT1A1.

DETAILED DESCRIPTION

It is of great interest in the fields of therapeutics, diagnostics,reagents and for biological assays to be able to deliver a nucleic acid,e.g., a ribonucleic acid (RNA) inside a cell, whether in vitro, in vivo,in situ or ex vivo, such as to cause intracellular translation of thenucleic acid and production of an encoded polypeptide of interest. Ofparticular importance is the delivery and function of a non-integrativepolynucleotide.

Described herein are compositions (including pharmaceuticalcompositions) and methods for the design, preparation, manufactureand/or formulation of polynucleotides encoding one or more polypeptidesof interest. Also provided are systems, processes, devices and kits forthe selection, design and/or utilization of the polynucleotides encodingthe polypeptides of interest described herein.

According to the present invention, these polynucleotides are preferablymodified as to avoid the deficiencies of other polypeptide-encodingmolecules of the art. Hence these polynucleotides are referred to asmodified mRNA or mmRNA.

The use of modified polynucleotides in the fields of antibodies,viruses, veterinary applications and a variety of in vivo settings hasbeen explored by the inventors and these studies are disclosed in forexample, co-pending and co-owned U.S. provisional patent applicationSer. Nos. 61/470,451 filed Mar. 31, 2011 teaching in vivo applicationsof mmRNA; 61/517,784 filed on Apr. 26, 2011 teaching engineered nucleicacids for the production of antibody polypeptides; 61/519,158 filed May17, 2011 teaching veterinary applications of mmRNA technology; 61/533,537 filed on Sep. 12, 2011 teaching antimicrobial applications of mmRNAtechnology; 61/533,554 filed on Sep. 12, 2011 teaching viralapplications of mmRNA technology, 61/542,533 filed on Oct. 3, 2011teaching various chemical modifications for use in mmRNA technology;61/570,690 filed on Dec. 14, 2011 teaching mobile devices for use inmaking or using mmRNA technology; 61/570,708 filed on Dec. 14, 2011teaching the use of mmRNA in acute care situations;

-   61/576,651 filed on Dec. 16, 2011 teaching terminal modification    architecture for mmRNA; 61/576,705 filed on Dec. 16, 2011 teaching    delivery methods using lipidoids for mmRNA; 61/578,271 filed on Dec.    21, 2011 teaching methods to increase the viability of organs or    tissues using mmRNA; 61/581,322 filed on Dec. 29, 2011 teaching    mmRNA encoding cell penetrating peptides; 61/581,352 filed on Dec.    29, 2011 teaching the incorporation of cytotoxic nucleosides in    mmRNA and 61/631,729 filed on Jan. 10, 2012 teaching methods of    using mmRNA for crossing the blood brain barrier; all of which are    herein incorporated by reference in their entirety.

Provided herein, in part, are polynucleotides, primary constructs and/ormmRNA encoding polypeptides of interest which have been designed toimprove one or more of the stability and/or clearance in tissues,receptor uptake and/or kinetics, cellular access by the compositions,engagement with translational machinery, mRNA half-life, translationefficiency, immune evasion, protein production capacity, secretionefficiency (when applicable), accessibility to circulation, proteinhalf-life and/or modulation of a cell's status, function and/oractivity.

I. Compositions of the Invention (mmRNA)

The present invention provides nucleic acid molecules, specificallypolynucleotides, primary constructs and/or mmRNA which encode one ormore polypeptides of interest. The term “nucleic acid,” in its broadestsense, includes any compound and/or substance that comprise a polymer ofnucleotides. These polymers are often referred to as polynucleotides.Exemplary nucleic acids or polynucleotides of the invention include, butare not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNAhaving a β-D-ribo configuration, α-LNA having an α-L-ribo configuration(a diastereomer of LNA), 2′-amino-LNA having a 2′-aminofunctionalization, and 2′-amino- α-LNA having a 2′-aminofunctionalization) or hybrids thereof.

In preferred embodiments, the nucleic acid molecule is a messenger RNA(mRNA). As used herein, the term “messenger RNA” (mRNA) refers to anypolynucleotide which encodes a polypeptide of interest and which iscapable of being translated to produce the encoded polypeptide ofinterest in vitro, in vivo, in situ or ex vivo.

Traditionally, the basic components of an mRNA molecule include at leasta coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail. Buildingon this wild type modular structure, the present invention expands thescope of functionality of traditional mRNA molecules by providingpolynucleotides or primary RNA constructs which maintain a modularorganization, but which comprise one or more structural and/or chemicalmodifications or alterations which impart useful properties to thepolynucleotide including, in some embodiments, the lack of a substantialinduction of the innate immune response of a cell into which thepolynucleotide is introduced. As such, modified mRNA molecules of thepresent invention are termed “mmRNA.” As used herein, a “structural”feature or modification is one in which two or more linked nucleotidesare inserted, deleted, duplicated, inverted or randomized in apolynucleotide, primary construct or mmRNA without significant chemicalmodification to the nucleotides themselves. Because chemical bonds willnecessarily be broken and reformed to effect a structural modification,structural modifications are of a chemical nature and hence are chemicalmodifications. However, structural modifications will result in adifferent sequence of nucleotides. For example, the polynucleotide“ATCG” may be chemically modified to “AT-5meC-G”. The samepolynucleotide may be structurally modified from “ATCG” to “ATCCCG”.Here, the dinucleotide “CC” has been inserted, resulting in a structuralmodification to the polynucleotide.

mmRNA Architecture

The mmRNA of the present invention are distinguished from wild type mRNAin their functional and/or structural design features which serve to, asevidenced herein, overcome existing problems of effective polypeptideproduction using nucleic acid-based therapeutics.

FIG. 1 shows a representative polynucleotide primary construct 100 ofthe present invention. As used herein, the term “primary construct” or“primary mRNA construct” refers to a polynucleotide transcript whichencodes one or more polypeptides of interest and which retainssufficient structural and/or chemical features to allow the polypeptideof interest encoded therein to be translated. Primary constructs may bepolynucleotides of the invention. When structurally or chemicallymodified, the primary construct may be referred to as an mmRNA.

Returning to FIG. 1, the primary construct 100 here contains a firstregion of linked nucleotides 102 that is flanked by a first flankingregion 104 and a second flaking region 106. As used herein, the “firstregion” may be referred to as a “coding region” or “region encoding” orsimply the “first region.” This first region may include, but is notlimited to, the encoded polypeptide of interest. The polypeptide ofinterest may comprise at its 5′ terminus one or more signal sequencesencoded by a signal sequence region 103. The flanking region 104 maycomprise a region of linked nucleotides comprising one or more completeor incomplete 5′ UTRs sequences. The flanking region 104 may alsocomprise a 5′ terminal cap 108. The second flanking region 106 maycomprise a region of linked nucleotides comprising one or more completeor incomplete 3′ UTRs. The flanking region 106 may also comprise a 3′tailing sequence 110.

Bridging the 5′ terminus of the first region 102 and the first flankingregion 104 is a first operational region 105. Traditionally thisoperational region comprises a Start codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a Start codon.

Bridging the 3′ terminus of the first region 102 and the second flankingregion 106 is a second operational region 107. Traditionally thisoperational region comprises a Stop codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a Stop codon. According to the present invention, multipleserial stop codons may also be used.

Generally, the shortest length of the first region of the primaryconstruct of the present invention can be the length of a nucleic acidsequence that is sufficient to encode for a dipeptide, a tripeptide, atetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, anoctapeptide, a nonapeptide, or a decapeptide. In another embodiment, thelength may be sufficient to encode a peptide of 2-30 amino acids, e.g.5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may besufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17,20, 25 or 30 amino acids, or a peptide that is no longer than 40 aminoacids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10amino acids. Examples of dipeptides that the polynucleotide sequencescan encode or include, but are not limited to, carnosine and anserine.

Generally, the length of the first region encoding the polypeptide ofinterest of the present invention is greater than about 30 nucleotidesin length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000,7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). Asused herein, the “first region” may be referred to as a “coding region”or “region encoding” or simply the “first region.”

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes from about 30 to about 100,000 nucleotides (e.g., from 30 to50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000,from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000,from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000,from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000,from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000,from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to70,000, and from 2,000 to 100,000).

According to the present invention, the first and second flankingregions may range independently from 15-1,000 nucleotides in length(e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,and 1,000 nucleotides).

According to the present invention, the tailing sequence may range fromabsent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Wherethe tailing region is a polyA tail, the length may be determined inunits of or as a function of polyA Binding Protein binding. In thisembodiment, the polyA tail is long enough to bind at least 4 monomers ofPolyA Binding Protein. PolyA Binding Protein monomers bind to stretchesof approximately 38 nucleotides. As such, it has been observed thatpolyA tails of about 80 nucleotides and 160 nucleotides are functional.

According to the present invention, the capping region may comprise asingle cap or a series of nucleotides forming the cap. In thisembodiment the capping region may be from 1 to 10, e.g. 2-9, 3-8, 4-7,1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In someembodiments, the cap is absent.

According to the present invention, the first and second operationalregions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or30 or fewer nucleotides in length and may comprise, in addition to aStart and/or Stop codon, one or more signal and/or restrictionsequences.

Cyclic mmRNA

According to the present invention, a primary construct or mmRNA may becyclized, or concatemerized, to generate a translation competentmolecule to assist interactions between poly-A binding proteins and5′-end binding proteins. The mechanism of cyclization orconcatemerization may occur through at least 3 different routes: 1)chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed5′-/3′-linkage may be intramolecular or intermolecular.

In the first route, the 5′-end and the 3′-end of the nucleic acidcontain chemically reactive groups that, when close together, form a newcovalent linkage between the 5′-end and the 3′-end of the molecule. The5′-end may contain an NHS-ester reactive group and the 3′-end maycontain a 3′-amino-terminated nucleotide such that in an organic solventthe 3′-amino-terminated nucleotide on the 3′-end of a synthetic mRNAmolecule will undergo a nucleophilic attack on the 5′—NHS-ester moietyforming a new 5′-/3′-amide bond.

In the second route, T4 RNA ligase may be used to enzymatically link a5′-phosphorylated nucleic acid molecule to the 3′-hydroxyl group of anucleic acid forming a new phosphorodiester linkage. In an examplereaction, 1 μg of a nucleic acid molecule is incubated at 37° C. for 1hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich,Mass.) according to the manufacturer's protocol. The ligation reactionmay occur in the presence of a split oligonucleotide capable ofbase-pairing with both the 5′- and 3′-region in juxtaposition to assistthe enzymatic ligation reaction.

In the third route, either the 5′- or 3′-end of the cDNA templateencodes a ligase ribozyme sequence such that during in vitrotranscription, the resultant nucleic acid molecule can contain an activeribozyme sequence capable of ligating the 5′-end of a nucleic acidmolecule to the 3′-end of a nucleic acid molecule. The ligase ribozymemay be derived from the Group I Intron, Group I Intron, Hepatitis DeltaVirus, Hairpin ribozyme or may be selected by SELEX (systematicevolution of ligands by exponential enrichment). The ribozyme ligasereaction may take 1 to 24 hours at temperatures between 0 and 37° C.

mmRNA Multimers

According to the present invention, multiple distinct polynucleotides,primary constructs or mmRNA may be linked together through the 3′-endusing nucleotides which are modified at the 3′-terminus. Chemicalconjugation may be used to control the stoichiometry of delivery intocells. For example, the glyoxylate cycle enzymes, isocitrate lyase andmalate synthase, may be supplied into HepG2 cells at a 1:1 ratio toalter cellular fatty acid metabolism. This ratio may be controlled bychemically linking polynucleotides, primary constructs or mmRNA using a3′-azido terminated nucleotide on one polynucleotide, primary constructor mmRNA species and a C5-ethynyl or alkynyl-containing nucleotide onthe opposite polynucleotide, primary construct or mmRNA species. Themodified nucleotide is added post-transcriptionally using terminaltransferase (New England Biolabs, Ipswich, Mass.) according to themanufacturer's protocol. After the addition of the 3′-modifiednucleotide, the two polynucleotide, primary construct or mmRNA speciesmay be combined in an aqueous solution, in the presence or absence ofcopper, to form a new covalent linkage via a click chemistry mechanismas described in the literature.

In another example, more than two polynucleotides may be linked togetherusing a functionalized linker molecule. For example, a functionalizedsaccharide molecule may be chemically modified to contain multiplechemical reactive groups (SH—, NH₂—, N₃, etc. . . . ) to react with thecognate moiety on a 3′-functionalized mRNA molecule (i.e., a3′-maleimide ester, 3′—NHS-ester, alkynyl). The number of reactivegroups on the modified saccharide can be controlled in a stoichiometricfashion to directly control the stoichiometric ratio of conjugatedpolynucleotide, primary construct or mmRNA.

mmRNA Conjugates and Combinations

In order to further enhance protein production, primary constructs ormmRNA of the present invention can be designed to be conjugated to otherpolynucleotides, dyes, intercalating agents (e.g. acridines),cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K),MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeledmarkers, enzymes, haptens (e.g. biotin), transport/absorptionfacilitators (e.g., aspirin, vitamin E, folic acid), syntheticribonucleases, proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell, hormones and hormone receptors,non-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, or a drug.

Conjugation may result in increased stability and/or half life and maybe particularly useful in targeting the polynucleotides, primaryconstructs or mmRNA to specific sites in the cell, tissue or organism.

According to the present invention, the mmRNA or primary constructs maybe administered with, or further encode one or more of RNAi agents,siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes,catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamersor vectors, and the like.

Bifunctional mmRNA

In one embodiment of the invention are bifunctional polynucleotides(e.g., bifunctional primary constructs or bifunctional mmRNA). As thename implies, bifunctional polynucleotides are those having or capableof at least two functions. These molecules may also by convention bereferred to as multi-functional.

The multiple functionalities of bifunctional polynucleotides may beencoded by the RNA (the function may not manifest until the encodedproduct is translated) or may be a property of the polynucleotideitself. It may be structural or chemical. Bifunctional modifiedpolynucleotides may comprise a function that is covalently orelectrostatically associated with the polynucleotides. Further, the twofunctions may be provided in the context of a complex of a mmRNA andanother molecule.

Bifunctional polynucleotides may encode peptides which areanti-proliferative. These peptides may be linear, cyclic, constrained orrandom coil. They may function as aptamers, signaling molecules, ligandsor mimics or mimetics thereof. Anti-proliferative peptides may, astranslated, be from 3 to 50 amino acids in length. They may be 5-40,10-30, or approximately 15 amino acids long. They may be single chain,multichain or branched and may form complexes, aggregates or anymulti-unit structure once translated.

Noncoding Polynucleotides and Primary Constructs

As described herein, provided are polynucleotides and primary constructshaving sequences that are partially or substantially not translatable,e.g., having a noncoding region. Such noncoding region may be the “firstregion” of the primary construct. Alternatively, the noncoding regionmay be a region other than the first region. Such molecules aregenerally not translated, but can exert an effect on protein productionby one or more of binding to and sequestering one or more translationalmachinery components such as a ribosomal protein or a transfer RNA(tRNA), thereby effectively reducing protein expression in the cell ormodulating one or more pathways or cascades in a cell which in turnalters protein levels. The polynucleotide or primary construct maycontain or encode one or more long noncoding RNA (lncRNA, or lincRNA) orportion thereof, a small nucleolar RNA (sno-RNA), micro RNA (miRNA),small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).

Polypeptides of Interest

According to the present invention, the primary construct is designed toencode one or more polypeptides of interest or fragments thereof. Apolypeptide of interest may include, but is not limited to, wholepolypeptides, a plurality of polypeptides or fragments of polypeptides,which independently may be encoded by one or more nucleic acids, aplurality of nucleic acids, fragments of nucleic acids or variants ofany of the aforementioned. As used herein, the term “polypeptides ofinterest” refer to any polypeptide which is selected to be encoded inthe primary construct of the present invention. As used herein,“polypeptide” means a polymer of amino acid residues (natural orunnatural) linked together most often by peptide bonds. The term, asused herein, refers to proteins, polypeptides, and peptides of any size,structure, or function. In some instances the polypeptide encoded issmaller than about 50 amino acids and the polypeptide is then termed apeptide. If the polypeptide is a peptide, it will be at least about 2,3, 4, or at least 5 amino acid residues long. Thus, polypeptides includegene products, naturally occurring polypeptides, synthetic polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide may be a singlemolecule or may be a multi-molecular complex such as a dimer, trimer ortetramer. They may also comprise single chain or multichain polypeptidessuch as antibodies or insulin and may be associated or linked. Mostcommonly disulfide linkages are found in multichain polypeptides. Theterm polypeptide may also apply to amino acid polymers in which one ormore amino acid residues are an artificial chemical analogue of acorresponding naturally occurring amino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity (homology) to a native or referencesequence, and preferably, they will be at least about 80%, morepreferably at least about 90% identical (homologous) to a native orreference sequence.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains one or more amino acids whichwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, e.g., phenylalanine may act as an inactivatingsubstitution for tyrosine; or alanine may act as an inactivatingsubstitution for serine.

“Homology” as it applies to amino acid sequences is defined as thepercentage of residues in the candidate amino acid sequence that areidentical with the residues in the amino acid sequence of a secondsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. Methods and computerprograms for the alignment are well known in the art. It is understoodthat homology depends on a calculation of percent identity but maydiffer in value due to gaps and penalties introduced in the calculation.

By “homologs” as it applies to polypeptide sequences means thecorresponding sequence of other species having substantial identity to asecond sequence of a second species.

“Analogs” is meant to include polypeptide variants which differ by oneor more amino acid alterations, e.g., substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present invention contemplates several types of compositions whichare polypeptide based including variants and derivatives. These includesubstitutional, insertional, deletion and covalent variants andderivatives. The term “derivative” is used synonymously with the term“variant” but generally refers to a molecule that has been modifiedand/or changed in any way relative to a reference molecule or startingmolecule.

As such, mmRNA encoding polypeptides containing substitutions,insertions and/or additions, deletions and covalent modifications withrespect to reference sequences, in particular the polypeptide sequencesdisclosed herein, are included within the scope of this invention. Forexample, sequence tags or amino acids, such as one or more lysines, canbe added to the peptide sequences of the invention (e.g., at theN-terminal or C-terminal ends). Sequence tags can be used for peptidepurification or localization. Lysines can be used to increase peptidesolubility or to allow for biotinylation. Alternatively, amino acidresidues located at the carboxy and amino terminal regions of the aminoacid sequence of a peptide or protein may optionally be deletedproviding for truncated sequences. Certain amino acids (e.g., C-terminalor N-terminal residues) may alternatively be deleted depending on theuse of the sequence, as for example, expression of the sequence as partof a larger sequence which is soluble, or linked to a solid support.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. The substitutions may be single, where only one amino acid inthe molecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Insertional variants” when referring to polypeptides are those with oneor more amino acids inserted immediately adjacent to an amino acid at aparticular position in a native or starting sequence. “Immediatelyadjacent” to an amino acid means connected to either the alpha-carboxyor alpha-amino functional group of the amino acid.

“Deletional variants” when referring to polypeptides are those with oneor more amino acids in the native or starting amino acid sequenceremoved. Ordinarily, deletional variants will have one or more aminoacids deleted in a particular region of the molecule.

“Covalent derivatives” when referring to polypeptides includemodifications of a native or starting protein with an organicproteinaceous or non-proteinaceous derivatizing agent, and/orpost-translational modifications. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of theprotein with an organic derivatizing agent that is capable of reactingwith selected side-chains or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-proteinantibodies for immunoaffinity purification of the recombinantglycoprotein. Such modifications are within the ordinary skill in theart and are performed without undue experimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the polypeptides produced in accordancewith the present invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)).

“Features” when referring to polypeptides are defined as distinct aminoacid sequence-based components of a molecule. Features of thepolypeptides encoded by the mmRNA of the present invention includesurface manifestations, local conformational shape, folds, loops,half-loops, domains, half-domains, sites, termini or any combinationthereof.

As used herein when referring to polypeptides the term “surfacemanifestation” refers to a polypeptide based component of a proteinappearing on an outermost surface.

As used herein when referring to polypeptides the term “localconformational shape” means a polypeptide based structural manifestationof a protein which is located within a definable space of the protein.

As used herein when referring to polypeptides the term “fold” refers tothe resultant conformation of an amino acid sequence upon energyminimization. A fold may occur at the secondary or tertiary level of thefolding process. Examples of secondary level folds include beta sheetsand alpha helices. Examples of tertiary folds include domains andregions formed due to aggregation or separation of energetic forces.Regions formed in this way include hydrophobic and hydrophilic pockets,and the like.

As used herein the term “turn” as it relates to protein conformationmeans a bend which alters the direction of the backbone of a peptide orpolypeptide and may involve one, two, three or more amino acid residues.

As used herein when referring to polypeptides the term “loop” refers toa structural feature of a polypeptide which may serve to reverse thedirection of the backbone of a peptide or polypeptide. Where the loop isfound in a polypeptide and only alters the direction of the backbone, itmay comprise four or more amino acid residues. Oliva et al. haveidentified at least 5 classes of protein loops (J. Mol. Biol 266 (4):814-830; 1997). Loops may be open or closed. Closed loops or “cyclic”loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsbetween the bridging moieties. Such bridging moieties may comprise acysteine-cysteine bridge (Cys-Cys) typical in polypeptides havingdisulfide bridges or alternatively bridging moieties may be non-proteinbased such as the dibromozylyl agents used herein.

As used herein when referring to polypeptides the term “half-loop”refers to a portion of an identified loop having at least half thenumber of amino acid resides as the loop from which it is derived. It isunderstood that loops may not always contain an even number of aminoacid residues. Therefore, in those cases where a loop contains or isidentified to comprise an odd number of amino acids, a half-loop of theodd-numbered loop will comprise the whole number portion or next wholenumber portion of the loop (number of amino acids of the loop/2+/−0.5amino acids). For example, a loop identified as a 7 amino acid loopcould produce half-loops of 3 amino acids or 4 amino acids(7/2=3.5+/−0.5 being 3 or 4).

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the term “half-domain”means a portion of an identified domain having at least half the numberof amino acid resides as the domain from which it is derived. It isunderstood that domains may not always contain an even number of aminoacid residues. Therefore, in those cases where a domain contains or isidentified to comprise an odd number of amino acids, a half-domain ofthe odd-numbered domain will comprise the whole number portion or nextwhole number portion of the domain (number of amino acids of thedomain/2+/−0.5 amino acids). For example, a domain identified as a 7amino acid domain could produce half-domains of 3 amino acids or 4 aminoacids (7/2=3.5+/−0.5 being 3 or 4). It is also understood thatsub-domains may be identified within domains or half-domains, thesesubdomains possessing less than all of the structural or functionalproperties identified in the domains or half domains from which theywere derived. It is also understood that the amino acids that compriseany of the domain types herein need not be contiguous along the backboneof the polypeptide (i.e., nonadjacent amino acids may fold structurallyto produce a domain, half-domain or subdomain).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” A site represents aposition within a peptide or polypeptide that may be modified,manipulated, altered, derivatized or varied within the polypeptide basedmolecules of the present invention.

As used herein the terms “termini” or “terminus” when referring topolypeptides refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but may include additional amino acids in the terminalregions. The polypeptide based molecules of the present invention may becharacterized as having both an N-terminus (terminated by an amino acidwith a free amino group (NH2)) and a C-terminus (terminated by an aminoacid with a free carboxyl group (COOH)). Proteins of the invention arein some cases made up of multiple polypeptide chains brought together bydisulfide bonds or by non-covalent forces (multimers, oligomers). Thesesorts of proteins will have multiple N- and C-termini. Alternatively,the termini of the polypeptides may be modified such that they begin orend, as the case may be, with a non-polypeptide based moiety such as anorganic conjugate.

Once any of the features have been identified or defined as a desiredcomponent of a polypeptide to be encoded by the primary construct ormmRNA of the invention, any of several manipulations and/ormodifications of these features may be performed by moving, swapping,inverting, deleting, randomizing or duplicating. Furthermore, it isunderstood that manipulation of features may result in the same outcomeas a modification to the molecules of the invention. For example, amanipulation which involved deleting a domain would result in thealteration of the length of a molecule just as modification of a nucleicacid to encode less than a full length molecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to, site directed mutagenesis. Theresulting modified molecules may then be tested for activity using invitro or in vivo assays such as those described herein or any othersuitable screening assay known in the art.

According to the present invention, the polypeptides may comprise aconsensus sequence which is discovered through rounds ofexperimentation. As used herein a “consensus” sequence is a singlesequence which represents a collective population of sequences allowingfor variability at one or more sites.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest of this invention. Forexample, provided herein is any protein fragment (meaning a polypeptidesequence at least one amino acid residue shorter than a referencepolypeptide sequence but otherwise identical) of a reference protein 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids inlength. In another example, any protein that includes a stretch of about20, about 30, about 40, about 50, or about 100 amino acids which areabout 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, or about 100% identical to any of the sequences described hereincan be utilized in accordance with the invention. In certainembodiments, a polypeptide to be utilized in accordance with theinvention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided or referenced herein.

Encoded Polypeptides

The primary constructs or mmRNA of the present invention may be designedto encode polypeptides of interest selected from any of several targetcategories including, but not limited to, biologics, antibodies,vaccines, therapeutic proteins or peptides, cell penetrating peptides,secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletalproteins, intracellular membrane bound proteins, nuclear proteins,proteins associated with human disease, targeting moieties or thoseproteins encoded by the human genome for which no therapeutic indicationhas been identified but which nonetheless have utility in areas ofresearch and discovery.

In one embodiment primary constructs or mmRNA may encode variantpolypeptides which have a certain identity with a reference polypeptidesequence. As used herein, a “reference polypeptide sequence” refers to astarting polypeptide sequence. Reference sequences may be wild typesequences or any sequence to which reference is made in the design ofanother sequence. A “reference polypeptide sequence” may, e.g., be anyone of SEQ ID NOs: 769-1392 as disclosed herein, e.g., any of SEQ ID NOs769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782,783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796,797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810,811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824,825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838,839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852,853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866,867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880,881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894,895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908,909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922,923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936,937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950,951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964,965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978,979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992,993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005,1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017,1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029,1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041,1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053,1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065,1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077,1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089,1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101,1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113,1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125,1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137,1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149,1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161,1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173,1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185,1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197,1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209,1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221,1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233,1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245,1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257,1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269,1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281,1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293,1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305,1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317,1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329,1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341,1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353,1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365,1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377,1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389,1390, 1391, 1392.

The term “identity” as known in the art, refers to a relationshipbetween the sequences of two or more peptides, as determined bycomparing the sequences. In the art, identity also means the degree ofsequence relatedness between peptides, as determined by the number ofmatches between strings of two or more amino acid residues. Identitymeasures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Identity ofrelated peptides can be readily calculated by known methods. Suchmethods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

In some embodiments, the polypeptide variant may have the same or asimilar activity as the reference polypeptide. Alternatively, thevariant may have an altered activity (e.g., increased or decreased)relative to a reference polypeptide. Generally, variants of a particularpolynucleotide or polypeptide of the invention will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity tothat particular reference polynucleotide or polypeptide as determined bysequence alignment programs and parameters described herein and known tothose skilled in the art. Such tools for alignment include those of theBLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A.Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman(1997), “Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs”, Nucleic Acids Res. 25:3389-3402.) Other toolsare described herein, specifically in the definition of “Identity.”

Default parameters in the BLAST algorithm include, for example, anexpect threshold of 10, Word size of 28, Match/Mismatch Scores 1, −2,Gap costs Linear. Any filter can be applied as well as a selection forspecies specific repeats, e.g., Homo sapiens.

Biologics

The polynucleotides, primary constructs or mmRNA disclosed herein, mayencode one or more biologics. As used herein, a “biologic” is apolypeptide-based molecule produced by the methods provided herein andwhich may be used to treat, cure, mitigate, prevent, or diagnose aserious or life-threatening disease or medical condition. Biologics,according to the present invention include, but are not limited to,allergenic extracts (e.g. for allergy shots and tests), bloodcomponents, gene therapy products, human tissue or cellular productsused in transplantation, vaccines, monoclonal antibodies, cytokines,growth factors, enzymes, thrombolytics, and immunomodulators, amongothers.

According to the present invention, one or more biologics currentlybeing marketed or in development may be encoded by the polynucleotides,primary constructs or mmRNA of the present invention. While not wishingto be bound by theory, it is believed that incorporation of the encodingpolynucleotides of a known biologic into the primary constructs or mmRNAof the invention will result in improved therapeutic efficacy due atleast in part to the specificity, purity and/or selectivity of theconstruct designs.

Antibodies

The primary constructs or mmRNA disclosed herein, may encode one or moreantibodies or fragments thereof. The term “antibody” includes monoclonalantibodies (including full length antibodies which have animmunoglobulin Fc region), antibody compositions with polyepitopicspecificity, multispecific antibodies (e.g., bispecific antibodies,diabodies, and single-chain molecules), as well as antibody fragments.The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein. As used herein, the term “monoclonal antibody” refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity. Chimericantibodies of interest herein include, but are not limited to,“primatized” antibodies comprising variable domain antigen-bindingsequences derived from a non-human primate (e.g., Old World Monkey, Apeetc.) and human constant region sequences.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies; nanobodies; single-chainantibody molecules and multispecific antibodies formed from antibodyfragments.

Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM,may be encoded by the mmRNA of the invention, including the heavy chainsdesignated alpha, delta, epsilon, gamma and mu, respectively. Alsoincluded are polynucleotide sequences encoding the subclasses, gamma andmu. Hence any of the subclasses of antibodies may be encoded in part orin whole and include the following subclasses: IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2.

According to the present invention, one or more antibodies or fragmentscurrently being marketed or in development may be encoded by thepolynucleotides, primary constructs or mmRNA of the present invention.While not wishing to be bound by theory, it is believed thatincorporation into the primary constructs of the invention will resultin improved therapeutic efficacy due at least in part to thespecificity, purity and selectivity of the mmRNA designs.

Antibodies encoded in the polynucleotides, primary constructs or mmRNAof the invention may be utilized to treat conditions or diseases in manytherapeutic areas such as, but not limited to, blood, cardiovascular,CNS, poisoning (including antivenoms), dermatology, endocrinology,gastrointestinal, medical imaging, musculoskeletal, oncology,immunology, respiratory, sensory and anti-infective.

In one embodiment, primary constructs or mmRNA disclosed herein mayencode monoclonal antibodies and/or variants thereof. Variants ofantibodies may also include, but are not limited to, substitutionalvariants, conservative amino acid substitution, insertional variants,deletional variants and/or covalent derivatives. In one embodiment, theprimary construct and/or mmRNA disclosed herein may encode animmunoglobulin Fc region. In another embodiment, the primary constructsand/or mmRNA may encode a variant immunoglobulin Fc region. As anon-limiting example, the primary constructs and/or mmRNA may encode anantibody having a variant immunoglobulin Fc region as described in U.S.Pat. No. 8,217,147 herein incorporated by reference in its entirety.

Vaccines

The primary constructs or mmRNA disclosed herein, may encode one or morevaccines. As used herein, a “vaccine” is a biological preparation thatimproves immunity to a particular disease or infectious agent. Accordingto the present invention, one or more vaccines currently being marketedor in development may be encoded by the polynucleotides, primaryconstructs or mmRNA of the present invention. While not wishing to bebound by theory, it is believed that incorporation into the primaryconstructs or mmRNA of the invention will result in improved therapeuticefficacy due at least in part to the specificity, purity and selectivityof the construct designs.

Vaccines encoded in the polynucleotides, primary constructs or mmRNA ofthe invention may be utilized to treat conditions or diseases in manytherapeutic areas such as, but not limited to, cardiovascular, CNS,dermatology, endocrinology, oncology, immunology, respiratory, andanti-infective.

Therapeutic Proteins or Peptides

The primary constructs or mmRNA disclosed herein, may encode one or morevalidated or “in testing” therapeutic proteins or peptides.

According to the present invention, one or more therapeutic proteins orpeptides currently being marketed or in development may be encoded bythe polynucleotides, primary constructs or mmRNA of the presentinvention. While not wishing to be bound by theory, it is believed thatincorporation into the primary constructs or mmRNA of the invention willresult in improved therapeutic efficacy due at least in part to thespecificity, purity and selectivity of the construct designs.

Therapeutic proteins and peptides encoded in the polynucleotides,primary constructs or mmRNA of the invention may be utilized to treatconditions or diseases in many therapeutic areas such as, but notlimited to, blood, cardiovascular, CNS, poisoning (includingantivenoms), dermatology, endocrinology, genetic, genitourinary,gastrointestinal, musculoskeletal, oncology, and immunology,respiratory, sensory and anti-infective.

Cell-Penetrating Polypeptides

The primary constructs or mmRNA disclosed herein, may encode one or morecell-penetrating polypeptides. As used herein, “cell-penetratingpolypeptide” or CPP refers to a polypeptide which may facilitate thecellular uptake of molecules. A cell-penetrating polypeptide of thepresent invention may contain one or more detectable labels. Thepolypeptides may be partially labeled or completely labeled throughout.The polynucleotide, primary construct or mmRNA may encode the detectablelabel completely, partially or not at all. The cell-penetrating peptidemay also include a signal sequence. As used herein, a “signal sequence”refers to a sequence of amino acid residues bound at the amino terminusof a nascent protein during protein translation. The signal sequence maybe used to signal the secretion of the cell-penetrating polypeptide.

In one embodiment, the polynucleotides, primary constructs or mmRNA mayalso encode a fusion protein. The fusion protein may be created byoperably linking a charged protein to a therapeutic protein. As usedherein, “operably linked” refers to the therapeutic protein and thecharged protein being connected in such a way to permit the expressionof the complex when introduced into the cell. As used herein, “chargedprotein” refers to a protein that carries a positive, negative oroverall neutral electrical charge. Preferably, the therapeutic proteinmay be covalently linked to the charged protein in the formation of thefusion protein. The ratio of surface charge to total or surface aminoacids may be approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or0.9.

The cell-penetrating polypeptide encoded by the polynucleotides, primaryconstructs or mmRNA may form a complex after being translated. Thecomplex may comprise a charged protein linked, e.g. covalently linked,to the cell-penetrating polypeptide. “Therapeutic protein” refers to aprotein that, when administered to a cell has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

In one embodiment, the cell-penetrating polypeptide may comprise a firstdomain and a second domain. The first domain may comprise a superchargedpolypeptide. The second domain may comprise a protein-binding partner.As used herein, “protein-binding partner” includes, but is not limitedto, antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further comprise anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where the polynucleotide, primary construct or mmRNA may beintroduced. The cell-penetrating polypeptide may also be capable ofpenetrating the first cell.

In a further embodiment, the cell-penetrating polypeptide is capable ofpenetrating a second cell. The second cell may be from the same area asthe first cell, or it may be from a different area. The area mayinclude, but is not limited to, tissues and organs. The second cell mayalso be proximal or distal to the first cell.

In one embodiment, the polynucleotides, primary constructs or mmRNA mayencode a cell-penetrating polypeptide which may comprise aprotein-binding partner. The protein binding partner may include, but isnot limited to, an antibody, a supercharged antibody or a functionalfragment. The polynucleotides, primary constructs or mmRNA may beintroduced into the cell where a cell-penetrating polypeptide comprisingthe protein-binding partner is introduced.

Secreted Proteins

Human and other eukaryotic cells are subdivided by membranes into manyfunctionally distinct compartments. Each membrane-bounded compartment,or organelle, contains different proteins essential for the function ofthe organelle. The cell uses “sorting signals,” which are amino acidmotifs located within the protein, to target proteins to particularcellular organelles.

One type of sorting signal, called a signal sequence, a signal peptide,or a leader sequence, directs a class of proteins to an organelle calledthe endoplasmic reticulum (ER).

Proteins targeted to the ER by a signal sequence can be released intothe extracellular space as a secreted protein. Similarly, proteinsresiding on the cell membrane can also be secreted into theextracellular space by proteolytic cleavage of a “linker” holding theprotein to the membrane. While not wishing to be bound by theory, themolecules of the present invention may be used to exploit the cellulartrafficking described above. As such, in some embodiments of theinvention, polynucleotides, primary constructs or mmRNA are provided toexpress a secreted protein. The secreted proteins may be selected fromthose described herein or those in US Patent Publication, 20100255574,the contents of which are incorporated herein by reference in theirentirety.

In one embodiment, these may be used in the manufacture of largequantities of valuable human gene products.

Plasma Membrane Proteins

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a protein of the plasmamembrane.

Cytoplasmic or Cytoskeletal Proteins

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a cytoplasmic orcytoskeletal protein.

Intracellular Membrane Bound Proteins

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express an intracellular membranebound protein.

Nuclear Proteins

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a nuclear protein.

Proteins Associated with Human Disease

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a protein associated withhuman disease.

Miscellaneous Proteins

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a protein with a presentlyunknown therapeutic function.

Targeting Moieties

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a targeting moiety. Theseinclude a protein-binding partner or a receptor on the surface of thecell, which functions to target the cell to a specific tissue space orto interact with a specific moiety, either in vivo or in vitro. Suitableprotein-binding partners include, but are not limited to, antibodies andfunctional fragments thereof, scaffold proteins, or peptides.Additionally, polynucleotide, primary construct or mmRNA can be employedto direct the synthesis and extracellular localization of lipids,carbohydrates, or other biological moieties or biomolecules.

Polypeptide Libraries

In one embodiment, the polynucleotides, primary constructs or mmRNA maybe used to produce polypeptide libraries. These libraries may arise fromthe production of a population of polynucleotides, primary constructs ormmRNA, each containing various structural or chemical modificationdesigns. In this embodiment, a population of polynucleotides, primaryconstructs or mmRNA may comprise a plurality of encoded polypeptides,including but not limited to, an antibody or antibody fragment, proteinbinding partner, scaffold protein, and other polypeptides taught hereinor known in the art. In a preferred embodiment, the polynucleotides areprimary constructs of the present invention, including mmRNA which maybe suitable for direct introduction into a target cell or culture whichin turn may synthesize the encoded polypeptides.

In certain embodiments, multiple variants of a protein, each withdifferent amino acid modification(s), may be produced and tested todetermine the best variant in terms of pharmacokinetics, stability,biocompatibility, and/or biological activity, or a biophysical propertysuch as expression level. Such a library may contain 10, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or over 10⁹ possible variants (including, butnot limited to, substitutions, deletions of one or more residues, andinsertion of one or more residues).

Anti-Microbial and Anti-viral Polypeptides

The polynucleotides, primary constructs and mmRNA of the presentinvention may be designed to encode on or more antimicrobial peptides(AMP) or antiviral peptides (AVP). AMPs and AVPs have been isolated anddescribed from a wide range of animals such as, but not limited to,microorganisms, invertebrates, plants, amphibians, birds, fish, andmammals (Wang et al., Nucleic Acids Res. 2009; 37 (Databaseissue):D933-7). For example, anti-microbial polypeptides are describedin Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php; Wanget al., Nucleic Acids Res. 2009; 37 (Database issue):D933-7), CAMP:Collection of Anti-Microbial Peptides(http://www.bicnirrh.res.in/antimicrobial/); Thomas et al., NucleicAcids Res. 2010; 38 (Database issue):D774-80), U.S. Pat. No. 5,221,732,U.S. Pat. No. 5,447,914, U.S. Pat. No. 5,519,115, U.S. Pat. No.5,607,914, U.S. Pat. No. 5,714,577, U.S. Pat. No. 5,734,015, U.S. Pat.No. 5,798,336, U.S. Pat. No. 5,821,224, U.S. Pat. No. 5,849,490, U.S.Pat. No. 5,856,127, U.S. Pat. No. 5,905,187, U.S. Pat. No. 5,994,308,U.S. Pat. No. 5,998,374, U.S. Pat. No. 6,107,460, U.S. Pat. No.6,191,254, U.S. Pat. No. 6,211,148, U.S. Pat. No. 6,300,489, U.S. Pat.No. 6,329,504, U.S. Pat. No. 6,399,370, U.S. Pat. No. 6,476,189, U.S.Pat. No. 6,478,825, U.S. Pat. No. 6,492,328, U.S. Pat. No. 6,514,701,U.S. Pat. No. 6,573,361, U.S. Pat. No. 6,573,361, U.S. Pat. No.6,576,755, U.S. Pat. No. 6,605,698, U.S. Pat. No. 6,624,140, U.S. Pat.No. 6,638,531, U.S. Pat. No. 6,642,203, U.S. Pat. No. 6,653,280, U.S.Pat. No. 6,696,238, U.S. Pat. No. 6,727,066, U.S. Pat. No. 6,730,659,U.S. Pat. No. 6,743,598, U.S. Pat. No. 6,743,769, U.S. Pat. No.6,747,007, U.S. Pat. No. 6,790,833, U.S. Pat. No. 6,794,490, U.S. Pat.No. 6,818,407, U.S. Pat. No. 6,835,536, U.S. Pat. No. 6,835,713, U.S.Pat. No. 6,838,435, U.S. Pat. No. 6,872,705, U.S. Pat. No. 6,875,907,U.S. Pat. No. 6,884,776, U.S. Pat. No. 6,887,847, U.S. Pat. No.6,906,035, U.S. Pat. No. 6,911,524, U.S. Pat. No. 6,936,432, U.S. Pat.No. 7,001,924, U.S. Pat. No. 7,071,293, U.S. Pat. No. 7,078,380, U.S.Pat. No. 7,091,185, U.S. Pat. No. 7,094,759, U.S. Pat. No. 7,166,769,U.S. Pat. No. 7,244,710, U.S. Pat. No. 7,314,858, and U.S. Pat. No.7,582,301, the contents of which are incorporated by reference in theirentirety.

The anti-microbial polypeptides described herein may block cell fusionand/or viral entry by one or more enveloped viruses (e.g., HIV, HCV).For example, the anti-microbial polypeptide can comprise or consist of asynthetic peptide corresponding to a region, e.g., a consecutivesequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or60 amino acids of the transmembrane subunit of a viral envelope protein,e.g., HIV-1 gp120 or gp41. The amino acid and nucleotide sequences ofHIV-1 gp120 or gp41 are described in, e.g., Kuiken et al., (2008). “HIVSequence Compendium,” Los Alamos National Laboratory.

In some embodiments, the anti-microbial polypeptide may have at leastabout 75%, 80%, 85%, 90%, 95%, 100% sequence homology to thecorresponding viral protein sequence. In some embodiments, theanti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%,95%, or 100% sequence homology to the corresponding viral proteinsequence.

In other embodiments, the anti-microbial polypeptide may comprise orconsist of a synthetic peptide corresponding to a region, e.g., aconsecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, or 60 amino acids of the binding domain of a capsid bindingprotein. In some embodiments, the anti-microbial polypeptide may have atleast about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to thecorresponding sequence of the capsid binding protein.

The anti-microbial polypeptides described herein may block proteasedimerization and inhibit cleavage of viral proproteins (e.g., HIVGag-pol processing) into functional proteins thereby preventing releaseof one or more enveloped viruses (e.g., HIV, HCV). In some embodiments,the anti-microbial polypeptide may have at least about 75%, 80%, 85%,90%, 95%, 100% sequence homology to the corresponding viral proteinsequence.

In other embodiments, the anti-microbial polypeptide can comprise orconsist of a synthetic peptide corresponding to a region, e.g., aconsecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, or 60 amino acids of the binding domain of a proteasebinding protein. In some embodiments, the anti-microbial polypeptide mayhave at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology tothe corresponding sequence of the protease binding protein.

The anti-microbial polypeptides described herein can include an invitro-evolved polypeptide directed against a viral pathogen.

Anti-Microbial Polypeptides

Anti-microbial polypeptides (AMPs) are small peptides of variablelength, sequence and structure with broad spectrum activity against awide range of microorganisms including, but not limited to, bacteria,viruses, fungi, protozoa, parasites, prions, and tumor/cancer cells.(See, e.g., Zaiou, J Mol Med, 2007; 85:317; herein incorporated byreference in its entirety). It has been shown that AMPs havebroad-spectrum of rapid onset of killing activities, with potentiallylow levels of induced resistance and concomitant broad anti-inflammatoryeffects.

In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be under 10 kDa, e.g., under 8 kDa, 6kDa, 4 kDa, 2 kDa, or 1 kDa. In some embodiments, the anti-microbialpolypeptide (e.g., an anti-bacterial polypeptide) consists of from about6 to about 100 amino acids, e.g., from about 6 to about 75 amino acids,about 6 to about 50 amino acids, about 6 to about 25 amino acids, about25 to about 100 amino acids, about 50 to about 100 amino acids, or about75 to about 100 amino acids. In certain embodiments, the anti-microbialpolypeptide (e.g., an anti-bacterial polypeptide) may consist of fromabout 15 to about 45 amino acids. In some embodiments, theanti-microbial polypeptide (e.g., an anti-bacterial polypeptide) issubstantially cationic.

In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be substantially amphipathic. In certainembodiments, the anti-microbial polypeptide (e.g., an anti-bacterialpolypeptide) may be substantially cationic and amphipathic. In someembodiments, the anti-microbial polypeptide (e.g., an anti-bacterialpolypeptide) may be cytostatic to a Gram-positive bacterium. In someembodiments, the anti-microbial polypeptide (e.g., an anti-bacterialpolypeptide) may be cytotoxic to a Gram-positive bacterium. In someembodiments, the anti-microbial polypeptide (e.g., an anti-bacterialpolypeptide) may be cytostatic and cytotoxic to a Gram-positivebacterium. In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be cytostatic to a Gram-negativebacterium. In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be cytotoxic to a Gram-negativebacterium. In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be cytostatic and cytotoxic to aGram-positive bacterium. In some embodiments, the anti-microbialpolypeptide may be cytostatic to a virus, fungus, protozoan, parasite,prion, or a combination thereof. In some embodiments, the anti-microbialpolypeptide may be cytotoxic to a virus, fungus, protozoan, parasite,prion, or a combination thereof. In certain embodiments, theanti-microbial polypeptide may be cytostatic and cytotoxic to a virus,fungus, protozoan, parasite, prion, or a combination thereof. In someembodiments, the anti-microbial polypeptide may be cytotoxic to a tumoror cancer cell (e.g., a human tumor and/or cancer cell). In someembodiments, the anti-microbial polypeptide may be cytostatic to a tumoror cancer cell (e.g., a human tumor and/or cancer cell). In certainembodiments, the anti-microbial polypeptide may be cytotoxic andcytostatic to a tumor or cancer cell (e.g., a human tumor or cancercell). In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be a secreted polypeptide.

In some embodiments, the anti-microbial polypeptide comprises orconsists of a defensin. Exemplary defensins include, but are not limitedto, α-defensins (e.g., neutrophil defensin 1, defensin alpha 1,neutrophil defensin 3, neutrophil defensin 4, defensin 5, defensin 6),β-defensins (e.g., beta-defensin 1, beta-defensin 2, beta-defensin 103,beta-defensin 107, beta-defensin 110, beta-defensin 136), andθ-defensins. In other embodiments, the anti-microbial polypeptidecomprises or consists of a cathelicidin (e.g., hCAP18).

Anti-Viral Polypeptides

Anti-viral polypeptides (AVPs) are small peptides of variable length,sequence and structure with broad spectrum activity against a wide rangeof viruses. See, e.g., Zaiou, J Mol Med, 2007; 85:317. It has been shownthat AVPs have a broad-spectrum of rapid onset of killing activities,with potentially low levels of induced resistance and concomitant broadanti-inflammatory effects. In some embodiments, the anti-viralpolypeptide is under 10 kDa, e.g., under 8 kDa, 6 kDa, 4 kDa, 2 kDa, or1 kDa. In some embodiments, the anti-viral polypeptide comprises orconsists of from about 6 to about 100 amino acids, e.g., from about 6 toabout 75 amino acids, about 6 to about 50 amino acids, about 6 to about25 amino acids, about 25 to about 100 amino acids, about 50 to about 100amino acids, or about 75 to about 100 amino acids. In certainembodiments, the anti-viral polypeptide comprises or consists of fromabout 15 to about 45 amino acids. In some embodiments, the anti-viralpolypeptide is substantially cationic. In some embodiments, theanti-viral polypeptide is substantially amphipathic. In certainembodiments, the anti-viral polypeptide is substantially cationic andamphipathic. In some embodiments, the anti-viral polypeptide iscytostatic to a virus. In some embodiments, the anti-viral polypeptideis cytotoxic to a virus. In some embodiments, the anti-viral polypeptideis cytostatic and cytotoxic to a virus. In some embodiments, theanti-viral polypeptide is cytostatic to a bacterium, fungus, protozoan,parasite, prion, or a combination thereof. In some embodiments, theanti-viral polypeptide is cytotoxic to a bacterium, fungus, protozoan,parasite, prion or a combination thereof. In certain embodiments, theanti-viral polypeptide is cytostatic and cytotoxic to a bacterium,fungus, protozoan, parasite, prion, or a combination thereof. In someembodiments, the anti-viral polypeptide is cytotoxic to a tumor orcancer cell (e.g., a human cancer cell). In some embodiments, theanti-viral polypeptide is cytostatic to a tumor or cancer cell (e.g., ahuman cancer cell). In certain embodiments, the anti-viral polypeptideis cytotoxic and cytostatic to a tumor or cancer cell (e.g., a humancancer cell). In some embodiments, the anti-viral polypeptide is asecreted polypeptide.

Cytotoxic Nucleosides

In one embodiment, the polynucleotides, primary constructs or mmRNA ofthe present invention may incorporate one or more cytotoxic nucleosides.For example, cytotoxic nucleosides may be incorporated intopolynucleotides, primary constructs or mmRNA such as bifunctionalmodified RNAs or mRNAs. Cytotoxic nucleoside anti-cancer agents include,but are not limited to, adenosine arabinoside, cytarabine, cytosinearabinoside, 5-fluorouracil, fludarabine, floxuridine, FTORAFUR® (acombination of tegafur and uracil), tegafur((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), and6-mercaptopurine.

A number of cytotoxic nucleoside analogues are in clinical use, or havebeen the subject of clinical trials, as anticancer agents. Examples ofsuch analogues include, but are not limited to, cytarabine, gemcitabine,troxacitabine, decitabine, tezacitabine, 2′-deoxy-2′-methylidenecytidine(DMDC), cladribine, clofarabine, 5-azacytidine, 4′-thio-aracytidine,cyclopentenylcytosine and1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine. Anotherexample of such a compound is fludarabine phosphate. These compounds maybe administered systemically and may have side effects which are typicalof cytotoxic agents such as, but not limited to, little or nospecificity for tumor cells over proliferating normal cells.

A number of prodrugs of cytotoxic nucleoside analogues are also reportedin the art. Examples include, but are not limited to,N4-behenoyl-1-beta-D-arabinofuranosylcytosine,N4-octadecyl-1-beta-D-arabinofuranosylcytosine,N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5′-elaidic acid ester). In general,these prodrugs may be converted into the active drugs mainly in theliver and systemic circulation and display little or no selectiverelease of active drug in the tumor tissue. For example, capecitabine, aprodrug of 5′-deoxy-5-fluorocytidine (and eventually of 5-fluorouracil),is metabolized both in the liver and in the tumor tissue. A series ofcapecitabine analogues containing “an easily hydrolysable radical underphysiological conditions” has been claimed by Fujiu et al. (U.S. Pat.No. 4,966,891) and is herein incorporated by reference. The seriesdescribed by Fujiu includes N4 alkyl and aralkyl carbamates of5′-deoxy-5-fluorocytidine and the implication that these compounds willbe activated by hydrolysis under normal physiological conditions toprovide 5′-deoxy-5-fluorocytidine.

A series of cytarabine N4-carbamates has been by reported by Fadl et al(Pharmazie. 1995, 50, 382-7, herein incorporated by reference) in whichcompounds were designed to convert into cytarabine in the liver andplasma. WO 2004/041203, herein incorporated by reference, disclosesprodrugs of gemcitabine, where some of the prodrugs are N4-carbamates.These compounds were designed to overcome the gastrointestinal toxicityof gemcitabine and were intended to provide gemcitabine by hydrolyticrelease in the liver and plasma after absorption of the intact prodrugfrom the gastrointestinal tract. Nomura et al (Bioorg Med. Chem. 2003,11, 2453-61, herein incorporated by reference) have described acetalderivatives of 1-β-C-ethynyl-β-D-ribo-pentofaranosyl) cytosine which, onbioreduction, produced an intermediate that required further hydrolysisunder acidic conditions to produce a cytotoxic nucleoside compound.

Cytotoxic nucleotides which may be chemotherapeutic also include, butare not limited to, pyrazolo[3,4-D]-pyrimidines, allopurinol,azathioprine, capecitabine, cytosine arabinoside, fluorouracil,mercaptopurine, 6-thioguanine, acyclovir, ara-adenosine, ribavirin,7-deaza-adenosine, 7-deaza-guanosine, 6-aza-uracil, 6-aza-cytidine,thymidine ribonucleotide, 5-bromodeoxyuridine, 2-chloro-purine, andinosine, or combinations thereof.

Flanking Regions Untranslated Regions (UTRs)

Untranslated regions (UTRs) of a gene are transcribed but nottranslated. The 5′UTR starts at the transcription start site andcontinues to the start codon but does not include the start codon;whereas, the 3′UTR starts immediately following the stop codon andcontinues until the transcriptional termination signal. There is growingbody of evidence about the regulatory roles played by the UTRs in termsof stability of the nucleic acid molecule and translation. Theregulatory features of a UTR can be incorporated into thepolynucleotides, primary constructs and/or mmRNA of the presentinvention to enhance the stability of the molecule. The specificfeatures can also be incorporated to ensure controlled down-regulationof the transcript in case they are misdirected to undesired organssites.

5′ UTR and Translation Initiation

Natural 5′UTRs bear features which play roles in for translationinitiation. They harbor signatures like Kozak sequences which arecommonly known to be involved in the process by which the ribosomeinitiates translation of many genes. Kozak sequences have the consensusCCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three basesupstream of the start codon (AUG), which is followed by another ‘G’.5′UTR also have been known to form secondary structures which areinvolved in elongation factor binding.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of the polynucleotides, primary constructs or mmRNAof the invention. For example, introduction of 5′ UTR of liver-expressedmRNA, such as albumin, serum amyloid A, Apolipoprotein A/B/E,transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, could beused to enhance expression of a nucleic acid molecule, such as a mmRNA,in hepatic cell lines or liver. Likewise, use of 5′ UTR from othertissue-specific mRNA to improve expression in that tissue is possiblefor muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), forendothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AML1, G-CSF,GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), foradipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lungepithelial cells (SP-A/B/C/D).

Other non-UTR sequences may be incorporated into the 5′ (or 3′ UTR)UTRs. For example, introns or portions of introns sequences may beincorporated into the flanking regions of the polynucleotides, primaryconstructs or mmRNA of the invention. Incorporation of intronicsequences may increase protein production as well as mRNA levels.

3′ UTR and the AU Rich Elements

UTRs are known to have stretches of Adenosines and Uridines embedded inthem. These AU rich signatures are particularly prevalent in genes withhigh rates of turnover. Based on their sequence features and functionalproperties, the AU rich elements (AREs) can be separated into threeclasses (Chen et al, 1995): Class I AREs contain several dispersedcopies of an AUUUA motif within U-rich regions. C-Myc and MyoD containclass I AREs. Class II AREs possess two or more overlappingUUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREsinclude GM-CSF and TNF-a. Class III ARES are less well defined. These Urich regions do not contain an AUUUA motif. c-Jun and Myogenin are twowell-studied examples of this class. Most proteins binding to the AREsare known to destabilize the messenger, whereas members of the ELAVfamily, most notably HuR, have been documented to increase the stabilityof mRNA. HuR binds to AREs of all the three classes. Engineering the HuRspecific binding sites into the 3′ UTR of nucleic acid molecules willlead to HuR binding and thus, stabilization of the message in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of polynucleotides, primaryconstructs or mmRNA of the invention. When engineering specificpolynucleotides, primary constructs or mmRNA, one or more copies of anARE can be introduced to make polynucleotides, primary constructs ormmRNA of the invention less stable and thereby curtail translation anddecrease production of the resultant protein. Likewise, AREs can beidentified and removed or mutated to increase the intracellularstability and thus increase translation and production of the resultantprotein. Transfection experiments can be conducted in relevant celllines, using polynucleotides, primary constructs or mmRNA of theinvention and protein production can be assayed at various time pointspost-transfection. For example, cells can be transfected with differentARE-engineering molecules and by using an ELISA kit to the relevantprotein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48hour, and 7 days post-transfection.

Incorporating microRNA Binding Sites

microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′UTR of nucleic acid molecules and down-regulate gene expressioneither by reducing nucleic acid molecule stability or by inhibitingtranslation. The polynucleotides, primary constructs or mmRNA of theinvention may comprise one or more microRNA target sequences, microRNAseqences, or microRNA seeds. Such sequences may correspond to any knownmicroRNA such as those taught in US Publication US2005/0261218 and USPublication US2005/0059005, the contents of which are incorporatedherein by reference in their entirety.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some embodiments, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked byan adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol. Cell. 2007 Jul. 6; 27(1):91-105; each of which isherein incorporated by reference in their entirety. The bases of themicroRNA seed have complete complementarity with the target sequence. Byengineering microRNA target sequences into the 3′UTR of polynucleotides,primary constructs or mmRNA of the invention one can target the moleculefor degradation or reduced translation, provided the microRNA inquestion is available. This process will reduce the hazard of off targeteffects upon nucleic acid molecule delivery. Identification of microRNA,microRNA target regions, and their expression patterns and role inbiology have been reported (Bonauer et al., Curr Drug Targets 201011:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176;Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20. doi:10.1038/leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al,Cell, 2007 129:1401-1414; each of which is herein incorporated byreference in its entirety).

For example, if the nucleic acid molecule is an mRNA and is not intendedto be delivered to the liver but ends up there, then miR-122, a microRNAabundant in liver, can inhibit the expression of the gene of interest ifone or multiple target sites of miR-122 are engineered into the 3′ UTRof the polynucleotides, primary constructs or mmRNA. Introduction of oneor multiple binding sites for different microRNA can be engineered tofurther decrease the longevity, stability, and protein translation of apolynucleotides, primary constructs or mmRNA.

As used herein, the term “microRNA site” refers to a microRNA targetsite or a microRNA recognition site, or any nucleotide sequence to whicha microRNA binds or associates. It should be understood that “binding”may follow traditional Watson-Crick hybridization rules or may reflectany stable association of the microRNA with the target sequence at oradjacent to the microRNA site.

Conversely, for the purposes of the polynucleotides, primary constructsor mmRNA of the present invention, microRNA binding sites can beengineered out of (i.e. removed from) sequences in which they naturallyoccur in order to increase protein expression in specific tissues. Forexample, miR-122 binding sites may be removed to improve proteinexpression in the liver. Regulation of expression in multiple tissuescan be accomplished through introduction or removal or one or severalmicroRNA binding sites.

Examples of tissues where microRNA are known to regulate mRNA, andthereby protein expression, include, but are not limited to, liver(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16,miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126). MicroRNA can also regulatecomplex biological processes such as angiogenesis (miR-132) (Anand andCheresh Curr Opin Hematol 2011 18:171-176; herein incorporated byreference in its entirety). In the polynucleotides, primary constructsor mmRNA of the present invention, binding sites for microRNAs that areinvolved in such processes may be removed or introduced, in order totailor the expression of the polynucleotides, primary constructs ormmRNA expression to biologically relevant cell types or to the contextof relevant biological processes. A listing of MicroRNA, miR sequencesand miR binding sites is listed in Table 9 of U.S. ProvisionalApplication No. 61/753,661 filed Jan. 17, 2013, in Table 9 of U.S.Provisional Application No. 61/754,159 filed Jan. 18, 2013, and in Table7 of U.S. Provisional Application No. 61/758,921 filed Jan. 31, 2013,each of which are herein incorporated by reference in their entireties.

Lastly, through an understanding of the expression patterns of microRNAin different cell types, polynucleotides, primary constructs or mmRNAcan be engineered for more targeted expression in specific cell types oronly under specific biological conditions. Through introduction oftissue-specific microRNA binding sites, polynucleotides, primaryconstructs or mmRNA could be designed that would be optimal for proteinexpression in a tissue or in the context of a biological condition.Examples of use of microRNA to drive tissue or disease-specific geneexpression are listed (Getner and Naldini, Tissue Antigens. 2012,80:393-403; herein incoroporated by reference in its entirety). Inaddition, microRNA seed sites can be incorporated into mRNA to decreaseexpression in certain cells which results in a biological improvement.An example of this is incorporation of miR-142 sites into aUGT1A1-expressing lentiviral vector. The presence of miR-142 seed sitesreduced expression in hematopoietic cells, and as a consequence reducedexpression in antigen-presentating cells, leading to the absence of animmune response against the virally expressed UGT1A1 (Schmitt et al.,Gastroenterology 2010; 139:999-1007; Gonzalez-Asequinolaza et al.Gastroenterology 2010, 139:726-729; both herein incorporated byreference in its entirety). Incorporation of miR-142 sites into modifiedmRNA could not only reduce expression of the encoded protein inhematopoietic cells, but could also reduce or abolish immune responsesto the mRNA-encoded protein. Incorporation of miR-142 seed sites (one ormultiple) into mRNA would be important in the case of treatment ofpatients with complete protein deficiencies (UGT1A1 type I,LDLR-deficient patients, CRIM-negative Pompe patients, etc.).

Transfection experiments can be conducted in relevant cell lines, usingengineered polynucleotides, primary constructs or mmRNA and proteinproduction can be assayed at various time points post-transfection. Forexample, cells can be transfected with different microRNA bindingsite-engineering polynucleotides, primary constructs or mmRNA and byusing an ELISA kit to the relevant protein and assaying protein producedat 6 hour, 12 hour, 24 hour, 48 hour, 72 hour and 7 dayspost-transfection. In vivo experiments can also be conducted usingmicroRNA-binding site-engineered molecules to examine changes intissue-specific expression of formulated polynucleotides, primaryconstructs or mmRNA.

5′ Capping

The 5′ cap structure of an mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsibile for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns removal during mRNA splicing.

Endogenous mRNA molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA mayoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

Modifications to the polynucleotides, primary constructs, and mmRNA ofthe present invention may generate a non-hydrolyzable cap structurepreventing decapping and thus increasing mRNA half-life. Because capstructure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiesterlinkages, modified nucleotides may be used during the capping reaction.For example, a Vaccinia Capping Enzyme from New England Biolabs(Ipswich, Mass.) may be used with α-thio-guanosine nucleotides accordingto the manufacturer's instructions to create a phosphorothioate linkagein the 5′-ppp-5′ cap. Additional modified guanosine nucleotides may beused such as α-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to,2′-O-methylation of the ribose sugars of 5′-terminal and/or5′-anteterminal nucleotides of the mRNA (as mentioned above) on the2′-hydroxyl group of the sugar ring. Multiple distinct 5′-cap structurescan be used to generate the 5′-cap of a nucleic acid molecule, such asan mRNA molecule.

Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e. endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs may be chemically (i.e. non-enzymatically) orenzymatically synthesized and/or linked to a nucleic acid molecule.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′ mppp-G;which may equivaliently be designated 3′ O-Me-m7G(5′)ppp(5′)G). The 3′-Oatom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped nucleic acid molecule (e.g. an mRNA or mmRNA).The N7- and 3′-O-methlyated guanine provides the terminal moiety of thecapped nucleic acid molecule (e.g. mRNA or mmRNA).

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

While cap analogs allow for the concomitant capping of a nucleic acidmolecule in an in vitro transcription reaction, up to 20% of transcriptscan remain uncapped. This, as well as the structural differences of acap analog from an endogenous 5′-cap structures of nucleic acidsproduced by the endogenous, cellular transcription machinery, may leadto reduced translational competency and reduced cellular stability.

Polynucleotides, primary constructs and mmRNA of the invention may alsobe capped post-transcriptionally, using enzymes, in order to generatemore authentic 5′-cap structures. As used herein, the phrase “moreauthentic” refers to a feature that closely mirrors or mimics, eitherstructurally or functionally, an endogenous or wild type feature. Thatis, a “more authentic” feature is better representative of anendogenous, wild-type, natural or physiological cellular function and/orstructure as compared to synthetic features or analogs, etc., of theprior art, or which outperforms the corresponding endogenous, wild-type,natural or physiological feature in one or more respects. Non-limitingexamples of more authentic 5′ cap structures of the present inventionare those which, among other things, have enhanced binding of capbinding proteins, increased half life, reduced susceptibility to 5′endonucleases and/or reduced 5′ decapping, as compared to synthetic 5′cap structures known in the art (or to a wild-type, natural orphysiological 5′ cap structure). For example, recombinant Vaccinia VirusCapping Enzyme and recombinant 2′-O-methyltransferase enzyme can createa canonical 5′-5′-triphosphate linkage between the 5′-terminalnucleotide of an mRNA and a guanine cap nucleotide wherein the capguanine contains an N7 methylation and the 5′-terminal nucleotide of themRNA contains a 2′-O-methyl. Such a structure is termed the Caplstructure. This cap results in a higher translational-competency andcellular stability and a reduced activation of cellular pro-inflammatorycytokines, as compared, e.g., to other 5′ cap analog structures known inthe art. Cap structures include, but are not limited to,7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)NlmpNp (cap 1), and7mG(5′)-ppp(5′)N1mpN2 mp (cap 2).

Because the polynucleotides, primary constructs or mmRNA may be cappedpost-transcriptionally, and because this process is more efficient,nearly 100% of the polynucleotides, primary constructs or mmRNA may becapped. This is in contrast to ˜80% when a cap analog is linked to anmRNA in the course of an in vitro transcription reaction.

According to the present invention, 5′ terminal caps may includeendogenous caps or cap analogs. According to the present invention, a 5′terminal cap may comprise a guanine analog. Useful guanine analogsinclude, but are not limited to, inosine, N1-methyl-guanosine,2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine,LNA-guanosine, and 2-azido-guanosine.

Viral Sequences

Additional viral sequences such as, but not limited to, the translationenhancer sequence of the barley yellow dwarf virus (BYDV-PAV), theJaagsiekte sheep retrovirus (JSRV) and/or the Enzootic nasal tumor virus(See e.g., International Pub. No. WO2012129648; herein incorporated byreference in its entirety) can be engineered and inserted in the 3′ UTRof the polynucleotides, primary constructs or mmRNA of the invention andcan stimulate the translation of the construct in vitro and in vivo.Transfection experiments can be conducted in relevant cell lines at andprotein production can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hrand day 7 post-transfection.

IRES Sequences

Further, provided are polynucleotides, primary constructs or mmRNA whichmay contain an internal ribosome entry site (IRES). First identified asa feature Picorna virus RNA, IRES plays an important role in initiatingprotein synthesis in absence of the 5′ cap structure. An IRES may act asthe sole ribosome binding site, or may serve as one of multiple ribosomebinding sites of an mRNA. Polynucleotides, primary constructs or mmRNAcontaining more than one functional ribosome binding site may encodeseveral peptides or polypeptides that are translated independently bythe ribosomes (“multicistronic nucleic acid molecules”). Whenpolynucleotides, primary constructs or mmRNA are provided with an IRES,further optionally provided is a second translatable region. Examples ofIRES sequences that can be used according to the invention includewithout limitation, those from picornaviruses (e.g. FMDV), pest viruses(CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV),foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV),classical swine fever viruses (CSFV), murine leukemia virus (MLV),simian immune deficiency viruses (SIV) or cricket paralysis viruses(CrPV).

Poly-A Tails

During RNA processing, a long chain of adenine nucleotides (poly-A tail)may be added to a polynucleotide such as an mRNA molecules in order toincrease stability. Immediately after transcription, the 3′ end of thetranscript may be cleaved to free a 3′ hydroxyl. Then poly-A polymeraseadds a chain of adenine nucleotides to the RNA. The process, calledpolyadenylation, adds a poly-A tail that can be between, for example,approximately 100 and 250 residues long.

It has been discovered that unique poly-A tail lengths provide certainadvantages to the polynucleotides, primary constructs or mmRNA of thepresent invention.

Generally, the length of a poly-A tail of the present invention isgreater than 30 nucleotides in length. In another embodiment, the poly-Atail is greater than 35 nucleotides in length (e.g., at least or greaterthan about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500,and 3,000 nucleotides). In some embodiments, the polynucleotide, primaryconstruct, or mmRNA includes from about 30 to about 3,000 nucleotides(e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500,from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000,from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500,from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000,from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

In one embodiment, the poly-A tail is designed relative to the length ofthe overall polynucleotides, primary constructs or mmRNA. This designmay be based on the length of the coding region, the length of aparticular feature or region (such as the first or flanking regions), orbased on the length of the ultimate product expressed from thepolynucleotides, primary constructs or mmRNA.

In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the polynucleotides, primaryconstructs or mmRNA or feature thereof. The poly-A tail may also bedesigned as a fraction of polynucleotides, primary constructs or mmRNAto which it belongs. In this context, the poly-A tail may be 10, 20, 30,40, 50, 60, 70, 80, or 90% or more of the total length of the constructor the total length of the construct minus the poly-A tail. Further,engineered binding sites and conjugation of polynucleotides, primaryconstructs or mmRNA for Poly-A binding protein may enhance expression.

Additionally, multiple distinct polynucleotides, primary constructs ormmRNA may be linked together to the PABP (Poly-A binding protein)through the 3′-end using modified nucleotides at the 3′-terminus of thepoly-A tail. Transfection experiments can be conducted in relevant celllines at and protein production can be assayed by ELISA at 12 hr, 24 hr,48 hr, 72 hr and day 7 post-transfection.

In one embodiment, the polynucleotide primary constructs of the presentinvention are designed to include a polyA-G Quartet. The G-quartet is acyclic hydrogen bonded array of four guanine nucleotides that can beformed by G-rich sequences in both DNA and RNA. In this embodiment, theG-quartet is incorporated at the end of the poly-A tail. The resultantmmRNA construct is assayed for stability, protein production and otherparameters including half-life at various time points. It has beendiscovered that the polyA-G quartet results in protein productionequivalent to at least 75% of that seen using a poly-A tail of 120nucleotides alone.

Quantification

In one embodiment, the polynucleotides, primary constructs or mmRNA ofthe present invention may be quantified in exosomes derived from one ormore bodily fluid. As used herein “bodily fluids” include peripheralblood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum,saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostaticfluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, and umbilical cord blood.Alternatively, exosomes may be retrieved from an organ selected from thegroup consisting of lung, heart, pancreas, stomach, intestine, bladder,kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus,liver, and placenta.

In the quantification method, a sample of not more than 2 mL is obtainedfrom the subject and the exosomes isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.In the analysis, the level or concentration of a polynucleotide, primaryconstruct or mmRNA may be an expression level, presence, absence,truncation or alteration of the administered construct. It isadvantageous to correlate the level with one or more clinical phenotypesor with an assay for a human disease biomarker. The assay may beperformed using construct specific probes, cytometry, qRT-PCR, real-timePCR, PCR, flow cytometry, electrophoresis, mass spectrometry, orcombinations thereof while the exosomes may be isolated usingimmunohistochemical methods such as enzyme linked immunosorbent assay(ELISA) methods. Exosomes may also be isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of polynucleotides, primary constructs or mmRNAremaining or delivered. This is possible because the polynucleotides,primary constructs or mmRNA of the present invention differ from theendogenous forms due to the structural or chemical modifications.

II. Design and Synthesis of mmRNA

Polynucleotides, primary constructs or mmRNA for use in accordance withthe invention may be prepared according to any available techniqueincluding, but not limited to chemical synthesis, enzymatic synthesis,which is generally termed in vitro transcription (IVT) or enzymatic orchemical cleavage of a longer precursor, etc. Methods of synthesizingRNAs are known in the art (see, e.g., Gait, M.J. (ed.) Oligonucleotidesynthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.:IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:methods and applications, Methods in Molecular Biology, v. 288 (Clifton,N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporatedherein by reference).

The process of design and synthesis of the primary constructs of theinvention generally includes the steps of gene construction, mRNAproduction (either with or without modifications) and purification. Inthe enzymatic synthesis method, a target polynucleotide sequenceencoding the polypeptide of interest is first selected for incorporationinto a vector which will be amplified to produce a cDNA template.Optionally, the target polynucleotide sequence and/or any flankingsequences may be codon optimized. The cDNA template is then used toproduce mRNA through in vitro transcription (IVT). After production, themRNA may undergo purification and clean-up processes. The steps of whichare provided in more detail below.

Gene Construction

The step of gene construction may include, but is not limited to genesynthesis, vector amplification, plasmid purification, plasmidlinearization and clean-up, and cDNA template synthesis and clean-up.

Gene Synthesis

Once a polypeptide of interest, or target, is selected for production, aprimary construct is designed. Within the primary construct, a firstregion of linked nucleosides encoding the polypeptide of interest may beconstructed using an open reading frame (ORF) of a selected nucleic acid(DNA or RNA) transcript. The ORF may comprise the wild type ORF, anisoform, variant or a fragment thereof. As used herein, an “open readingframe” or “ORF” is meant to refer to a nucleic acid sequence (DNA orRNA) which is capable of encoding a polypeptide of interest. ORFs oftenbegin with the start codon, ATG and end with a nonsense or terminationcodon or signal.

Further, the nucleotide sequence of the first region may be codonoptimized. Codon optimization methods are known in the art and may beuseful in efforts to achieve one or more of several goals. These goalsinclude to match codon frequencies in target and host organisms toensure proper folding, bias GC content to increase mRNA stability orreduce secondary structures, minimize tandem repeat codons or base runsthat may impair gene construction or expression, customizetranscriptional and translational control regions, insert or removeprotein trafficking sequences, remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modifyribosome binding sites and mRNA degradation sites, to adjusttranslational rates to allow the various domains of the protein to foldproperly, or to reduce or eliminate problem secondary structures withinthe mRNA. Codon optimization tools, algorithms and services are known inthe art, non-limiting examples include services from GeneArt (LifeTechnologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods. Inone embodiment, the ORF sequence is optimized using optimizationalgorithms. Codon options for each amino acid are given in Table 1.

TABLE 1 Codon Options Single Letter Amino Acid Code Codon OptionsIsoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA, CTG, TTA, TTG ValineV GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC Methionine M ATG CysteineC TGT, TGC Alanine A GCT, GCC, GCA, GCG Glycine G GGT, GGC, GGA, GGGProline P CCT, CCC, CCA, CCG Threonine T ACT, ACC, ACA, ACG Serine STCT, TCC, TCA, TCG, AGT, AGC Tyrosine Y TAT, TAC Tryptophan W TGGGlutamine Q CAA, CAG Asparagine N AAT, AAC Histidine H CAT, CAC Glutamicacid E GAA, GAG Aspartic acid D GAT, GAC Lysine K AAA, AAG Arginine RCGT, CGC, CGA, CGG, AGA, AGG Selenocysteine Sec UGA in mRNA in presenceof Selenocystein insertion element (SECIS) Stop codons Stop TAA, TAG,TGA

Features, which may be considered beneficial in some embodiments of thepresent invention, may be encoded by the primary construct and may flankthe ORF as a first or second flanking region. The flanking regions maybe incorporated into the primary construct before and/or afteroptimization of the ORF. It is not required that a primary constructcontain both a 5′ and 3′ flanking region. Examples of such featuresinclude, but are not limited to, untranslated regions (UTRs), Kozaksequences, an oligo(dT) sequence, and detectable tags and may includemultiple cloning sites which may have XbaI recognition.

In some embodiments, a 5′ UTR and/or a 3′ UTR may be provided asflanking regions. Multiple 5′ or 3′ UTRs may be included in the flankingregions and may be the same or of different sequences. Any portion ofthe flanking regions, including none, may be codon optimized and any mayindependently contain one or more different structural or chemicalmodifications, before and/or after codon optimization. Combinations offeatures may be included in the first and second flanking regions andmay be contained within other features. For example, the ORF may beflanked by a 5′ UTR which may contain a strong Kozak translationalinitiation signal and/or a 3′ UTR which may include an oligo(dT)sequence for templated addition of a poly-A tail. 5′UTR may comprise afirst polynucleotide fragment and a second polynucleotide fragment fromthe same and/or different genes such as the 5′UTRs described in USPatent Application Publication No. 20100293625, herein incorporated byreference in its entirety.

Tables 2 and 3 provide a listing of exemplary UTRs which may be utilizedin the primary construct of the present invention as flanking regions.Shown in Table 2 is a listing of a 5′-untranslated region of theinvention. Variants of 5′ UTRs may be utilized wherein one or morenucleotides are added or removed to the termini, including A, T, C or G.

TABLE 2 5′-Untranslated Regions 5′ UTR SEQ Identi- Name/ ID fierDescription Sequence NO 5UTR- Upstream UTR GGGAAATAAGAGAGAAAAGAAG 1 001AGTAAGAAGAAATATAAGAGCCA CC 5UTR- Upstream UTR GGGAGATCAGAGAGAAAAGAAG 2002 AGTAAGAAGAAATATAAGAGCCA CC 5UTR- Upstream UTRGGAATAAAAGTCTCAACACAACA 3 003 TATACAAAACAAACGAATCTCAAGCAATCAAGCATTCTACTTCTATT GCAGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAA ATTTTCACCATTTACGAACGATAG CAAC 5UTR- Upstream UTRGGGAGACAAGCUUGGCAUUCCG 4 004 GUACUGUUGGUAAAGCCACC

Shown in Table 3 is a representative listing of 3′-untranslated regionsof the invention. Variants of 3′ UTRs may be utilized wherein one ormore nucleotides are added or removed to the termini, including A, T, Cor G.

TABLE 3 3′-Untranslated Regions 3′ UTR SEQ ID IdentifierName/Description Sequence NO. 3UTR-001 CreatineGCGCCTGCCCACCTGCCACCGACTGCTGG 5 Kinase AACCCAGCCAGTGGGAGGGCCTGGCCCACCAGAGTCCTGCTCCCTCACTCCTCGCCCCG CCCCCTGTCCCAGAGTCCCACCTGGGGGCTCTCTCCACCCTTCTCAGAGTTCCAGTTTC AACAGAGTTCCAACCAATGGGCTCCATCCTCTGGATTCTGGCCAATGAAATATCTCCC TGGCAGGGTCCTCTTCTTTTCCCAGAGCTCCACCCCAACCAGGAGCTCTAGTTAATGGA GAGCTCCCAGCACACTCGGAGCTTGTGCTTTGTCTCCACGCAAAGCGATAAATAAAAG CATTGGTGGCCTTTGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR-002 Myoglobin GCCCCTGCCGCTCCCACCCCCACCCATCTG 6GGCCCCGGGTTCAAGAGAGAGCGGGGTCT GATCTCGTGTAGCCATATAGAGTTTGCTTCTGAGTGTCTGCTTTGTTTAGTAGAGGTGGG CAGGAGGAGCTGAGGGGCTGGGGCTGGGGTGTTGAAGTTGGCTTTGCATGCCCAGCG ATGCGCCTCCCTGTGGGATGTCATCACCCTGGGAACCGGGAGTGGCCCTTGGCTCACTG TGTTCTGCATGGTTTGGATCTGAATTAATTGTCCTTTCTTCTAAATCCCAACCGAACTTC TTCCAACCTCCAAACTGGCTGTAACCCCAAATCCAAGCCATTAACTACACCTGACAGT AGCAATTGTCTGATTAATCACTGGCCCCTTGAAGACAGCAGAATGTCCCTTTGCAATGA GGAGGAGATCTGGGCTGGGCGGGCCAGCTGGGGAAGCATTTGACTATCTGGAACTTGT GTGTGCCTCCTCAGGTATGGCAGTGACTCACCTGGTTTTAATAAAACAACCTGCAACA TCTCATGGTCTTTGAATAAAGCCTGAGTAG GAAGTCTAGA3UTR-003 α-actin ACACACTCCACCTCCAGCACGCGACTTCTC 7AGGACGACGAATCTTCTCAATGGGGGGGC GGCTGAGCTCCAGCCACCCCGCAGTCACTTTCTTTGTAACAACTTCCGTTGCTGCCATC GTAAACTGACACAGTGTTTATAACGTGTACATACATTAACTTATTACCTCATTTTGTTA TTTTTCGAAACAAAGCCCTGTGGAAGAAAATGGAAAACTTGAAGAAGCATTAAAGTCA TTCTGTTAAGCTGCGTAAATGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR-004 Albumin CATCACATTTAAAAGCATCTCAGCCTACC8 ATGAGAATAAGAGAAAGAAAATGAAGAT CAAAAGCTTATTCATCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAA AACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGG AAAGAATCTAATAGAGTGGTACAGCACTGTTATTTTTCAAAGATGTGTTGCTATCCTGA AAATTCTGTAGGTTCTGTGGAAGTTCCAGTGTTCTCTCTTATTCCACTTCGGTAGAGGAT TTCTAGTTTCTTGTGGGCTAATTAAATAAATCATTAATACTCTTCTAATGGTCTTTGAAT AAAGCCTGAGTAGGAAGTCTAGA 3UTR-005 α-globinGCTGCCTTCTGCGGGGCTTGCCTTCTGGCC 9 ATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGG AAGGCGGCCGCTCGAGCATGCATCTAGA 3UTR-006G-CSF GCCAAGCCCTCCCCATCCCATGTATTTATC 10 TCTATTTAATATTTATGTCTATTTAAGCCTCATATTTAAAGACAGGGAAGAGCAGAACG GAGCCCCAGGCCTCTGTGTCCTTCCCTGCATTTCTGAGTTTCATTCTCCTGCCTGTAGCA GTGAGAAAAAGCTCCTGTCCTCCCATCCCCTGGACTGGGAGGTAGATAGGTAAATACC AAGTATTTATTACTATGACTGCTCCCCAGCCCTGGCTCTGCAATGGGCACTGGGATGAG CCGCTGTGAGCCCCTGGTCCTGAGGGTCCCCACCTGGGACCCTTGAGAGTATCAGGTC TCCCACGTGGGAGACAAGAAATCCCTGTTTAATATTTAAACAGCAGTGTTCCCCATCTG GGTCCTTGCACCCCTCACTCTGGCCTCAGCCGACTGCACAGCGGCCCCTGCATCCCCTT GGCTGTGAGGCCCCTGGACAAGCAGAGGTGGCCAGAGCTGGGAGGCATGGCCCTGGGG TCCCACGAATTTGCTGGGGAATCTCGTTTTTCTTCTTAAGACTTTTGGGACATGGTTTGA CTCCCGAACATCACCGACGCGTCTCCTGTTTTTCTGGGTGGCCTCGGGACACCTGCCCTG CCCCCACGAGGGTCAGGACTGTGACTCTTTTTAGGGCCAGGCAGGTGCCTGGACATTT GCCTTGCTGGACGGGGACTGGGGATGTGGGAGGGAGCAGACAGGAGGAATCATGTCA GGCCTGTGTGTGAAAGGAAGCTCCACTGTCACCCTCCACCTCTTCACCCCCCACTCACC AGTGTCCCCTCCACTGTCACATTGTAACTGAACTTCAGGATAATAAAGTGTTTGCCTCC ATGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA 3UTR-007 Col1a2;ACTCAATCTAAATTAAAAAAGAAAGAAAT 11 collagen,TTGAAAAAACTTTCTCTTTGCCATTTCTTC type I, TTCTTCTTTTTTAACTGAAAGCTGAATCCTalpha 2 TCCATTTCTTCTGCACATCTACTTGCTTAA ATTGTGGGCAAAAGAGAAAAAGAAGGATTGATCAGAGCATTGTGCAATACAGTTTCAT TAACTCCTTCCCCCGCTCCCCCAAAAATTTGAATTTTTTTTTCAACACTCTTACACCTGT TATGGAAAATGTCAACCTTTGTAAGAAAACCAAAATAAAAATTGAAAAATAAAAACCA TAAACATTTGCACCACTTGTGGCTTTTGAATATCTTCCACAGAGGGAAGTTTAAAACCC AAACTTCCAAAGGTTTAAACTACCTCAAAACACTTTCCCATGAGTGTGATCCACATTGT TAGGTGCTGACCTAGACAGAGATGAACTGAGGTCCTTGTTTTGTTTTGTTCATAATACA AAGGTGCTAATTAATAGTATTTCAGATACTTGAAGAATGTTGATGGTGCTAGAAGAATT TGAGAAGAAATACTCCTGTATTGAGTTGTATCGTGTGGTGTATTTTTTAAAAAATTTGA TTTAGCATTCATATTTTCCATCTTATTCCCAATTAAAAGTATGCAGATTATTTGCCCAAA TCTTCTTCAGATTCAGCATTTGTTCTTTGCCAGTCTCATTTTCATCTTCTTCCATGGTTCC ACAGAAGCTTTGTTTCTTGGGCAAGCAGAAAAATTAAATTGTACCTATTTTGTATATGT GAGATGTTTAAATAAATTGTGAAAAAAATGAAATAAAGCATGTTTGGTTTTCCAAAAG AACATAT 3UTR-008 Col6a2;CGCCGCCGCCCGGGCCCCGCAGTCGAGGG 12 collagen, TCGTGAGCCCACCCCGTCCATGGTGCTAAtype VI, GCGGGCCCGGGTCCCACACGGCCAGCACC alpha 2GCTGCTCACTCGGACGACGCCCTGGGCCT GCACCTCTCCAGCTCCTCCCACGGGGTCCCCGTAGCCCCGGCCCCCGCCCAGCCCCAGG TCTCCCCAGGCCCTCCGCAGGCTGCCCGGCCTCCCTCCCCCTGCAGCCATCCCAAGGCT CCTGACCTACCTGGCCCCTGAGCTCTGGAGCAAGCCCTGACCCAATAAAGGCTTTGAA CCCAT 3UTR-009 RPN1;GGGGCTAGAGCCCTCTCCGCACAGCGTGG 13 ribophorin IAGACGGGGCAAGGAGGGGGGTTATTAGG ATTGGTGGTTTTGTTTTGCTTTGTTTAAAGCCGTGGGAAAATGGCACAACTTTACCTCT GTGGGAGATGCAACACTGAGAGCCAAGGGGTGGGAGTTGGGATAATTTTTATATAAA AGAAGTTTTTCCACTTTGAATTGCTAAAAGTGGCATTTTTCCTATGTGCAGTCACTCCTC TCATTTCTAAAATAGGGACGTGGCCAGGCACGGTGGCTCATGCCTGTAATCCCAGCAC TTTGGGAGGCCGAGGCAGGCGGCTCACGAGGTCAGGAGATCGAGACTATCCTGGCTAA CACGGTAAAACCCTGTCTCTACTAAAAGTACAAAAAATTAGCTGGGCGTGGTGGTGGG CACCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAAAGGCATGAATCCAAGA GGCAGAGCTTGCAGTGAGCTGAGATCACGCCATTGCACTCCAGCCTGGGCAACAGTGT TAAGACTCTGTCTCAAATATAAATAAATAAATAAATAAATAAATAAATAAATAAAAAT AAAGCGAGATGTTGCCCTCAAA 3UTR-010 LRP1;GGCCCTGCCCCGTCGGACTGCCCCCAGAA 14 low AGCCTCCTGCCCCCTGCCAGTGAAGTCCTTdensity CAGTGAGCCCCTCCCCAGCCAGCCCTTCCC lipoproteinTGGCCCCGCCGGATGTATAAATGTAAAAA receptor- TGAAGGAATTACATTTTATATGTGAGCGArelated GCAAGCCGGCAAGCGAGCACAGTATTATT protein 1TCTCCATCCCCTCCCTGCCTGCTCCTTGGC ACCCCCATGCTGCCTTCAGGGAGACAGGCAGGGAGGGCTTGGGGCTGCACCTCCTACC CTCCCACCAGAACGCACCCCACTGGGAGAGCTGGTGGTGCAGCCTTCCCCTCCCTGTAT AAGACACTTTGCCAAGGCTCTCCCCTCTCGCCCCATCCCTGCTTGCCCGCTCCCACAGCT TCCTGAGGGCTAATTCTGGGAAGGGAGAGTTCTTTGCTGCCCCTGTCTGGAAGACGTGG CTCTGGGTGAGGTAGGCGGGAAAGGATGGAGTGTTTTAGTTCTTGGGGGAGGCCACCCC AAACCCCAGCCCCAACTCCAGGGGCACCTATGAGATGGCCATGCTCAACCCCCCTCCC AGACAGGCCCTCCCTGTCTCCAGGGCCCCCACCGAGGTTCCCAGGGCTGGAGACTTCC TCTGGTAAACATTCCTCCAGCCTCCCCTCCCCTGGGGACGCCAAGGAGGTGGGCCACAC CCAGGAAGGGAAAGCGGGCAGCCCCGTTTTGGGGACGTGAACGTTTTAATAATTTTTGC TGAATTCCTTTACAACTAAATAACACAGATATTGTTATAAATAAAATTGT 3UTR-011 Nnt1; ATATTAAGGATCAAGCTGTTAGCTAATAA 15cardiotrophin- TGCCACCTCTGCAGTTTTGGGAACAGGCA likeAATAAAGTATCAGTATACATGGTGATGTA cytokine CATCTGTAGCAAAGCTCTTGGAGAAAATGfactor 1 AAGACTGAAGAAAGCAAAGCAAAAACTG TATAGAGAGATTTTTCAAAAGCAGTAATCCCTCAATTTTAAAAAAGGATTGAAAATTC TAAATGTCTTTCTGTGCATATTTTTTGTGTTAGGAATCAAAAGTATTTTATAAAAGGAGA AAGAACAGCCTCATTTTAGATGTAGTCCTGTTGGATTTTTTATGCCTCCTCAGTAACCA GAAATGTTTTAAAAAACTAAGTGTTTAGGATTTCAAGACAACATTATACATGGCTCTG AAATATCTGACACAATGTAAACATTGCAGGCACCTGCATTTTATGTTTTTTTTTTCAACA AATGTGACTAATTTGAAACTTTTATGAACTTCTGAGCTGTCCCCTTGCAATTCAACCGCA GTTTGAATTAATCATATCAAATCAGTTTTAATTTTTTAAATTGTACTTCAGAGTCTATAT TTCAAGGGCACATTTTCTCACTACTATTTTAATACATTAAAGGACTAAATAATCTTTCA GAGATGCTGGAAACAAATCATTTGCTTTATATGTTTCATTAGAATACCAATGAAACAT ACAACTTGAAAATTAGTAATAGTATTTTTGAAGATCCCATTTCTAATTGGAGATCTCTTT AATTTCGATCAACTTATAATGTGTAGTACTATATTAAGTGCACTTGAGTGGAATTCAAC ATTTGACTAATAAAATGAGTTCATCATGTTGGCAAGTGATGTGGCAATTATCTCTGGTG ACAAAAGAGTAAAATCAAATATTTCTGCCTGTTACAAATATCAAGGAAGACCTGCTAC TATGAAATAGATGACATTAATCTGTCTTCACTGTTTATAATACGGATGGATTTTTTTTCA AATCAGTGTGTGTTTTGAGGTCTTATGTAATTGATGACATTTGAGAGAAATGGTGGCTT TTTTTAGCTACCTCTTTGTTCATTTAAGCACCAGTAAAGATCATGTCTTTTTATAGAAGT GTAGATTTTCTTTGTGACTTTGCTATCGTGCCTAAAGCTCTAAATATAGGTGAATGTGT GATGAATACTCAGATTATTTGTCTCTCTATATAATTAGTTTGGTACTAAGTTTCTCAAAA AATTATTAACACATGAAAGACAATCTCTAAACCAGAAAAAGAAGTAGTACAAATTTTG TTACTGTAATGCTCGCGTTTAGTGAGTTTAAAACACACAGTATCTTTTGGTTTTATAATC AGTTTCTATTTTGCTGTGCCTGAGATTAAGATCTGTGTATGTGTGTGTGTGTGTGTGTGC GTTTGTGTGTTAAAGCAGAAAAGACTTTTTTAAAAGTTTTAAGTGATAAATGCAATTTGT TAATTGATCTTAGATCACTAGTAAACTCAGGGCTGAATTATACCATGTATATTCTATTAG AAGAAAGTAAACACCATCTTTATTCCTGCCCTTTTTCTTCTCTCAAAGTAGTTGTAGTT ATATCTAGAAAGAAGCAATTTTGATTTCTTGAAAAGGTAGTTCCTGCACTCAGTTTAAA CTAAAAATAATCATACTTGGATTTTATTTATTTTTGTCATAGTAAAAATTTTAATTTATA TATATTTTTATTTAGTATTATCTTATTCTTTGCTATTTGCCAATCCTTTGTCATCAATTGT GTTAAATGAATTGAAAATTCATGCCCTGTTCATTTTATTTTACTTTATTGGTTAGGATATT TAAAGGATTTTTGTATATATAATTTCTTAAATTAATATTCCAAAAGGTTAGTGGACTTA GATTATAAATTATGGCAAAAATCTAAAAACAACAAAAATGATTTTTATACATTCTATTT CATTATTCCTCTTTTTCCAATAAGTCATACAATTGGTAGATATGACTTATTTTATTTTTG TATTATTCACTATATCTTTATGATATTTAAGTATAAATAATTAAAAAAATTTATTGTAC CTTATAGTCTGTCACCAAAAAAAAAAAATTATCTGTAGGTAGTGAAATGCTAATGTTG ATTTGTCTTTAAGGGCTTGTTAACTATCCTTTATTTTCTCATTTGTCTTAAATTAGGAGT TTGTGTTTAAATTACTCATCTAAGCAAAAAATGTATATAAATCCCATTACTGGGTATATA CCCAAAGGATTATAAATCATGCTGCTATAAAGACACATGCACACGTATGTTTATTGCA GCACTATTCACAATAGCAAAGACTTGGAACCAACCCAAATGTCCATCAATGATAGACT TGATTAAGAAAATGTGCACATATACACCATGGAATACTATGCAGCCATAAAAAAGGAT GAGTTCATGTCCTTTGTAGGGACATGGATAAAGCTGGAAACCATCATTCTGAGCAAAC TATTGCAAGGACAGAAAACCAAACACTGCATGTTCTCACTCATAGGTGGGAATTGAAC AATGAGAACACTTGGACACAAGGTGGGGAACACCACACACCAGGGCCTGTCATGGGGT GGGGGGAGTGGGGAGGGATAGCATTAGGAGATATACCTAATGTAAATGATGAGTTAA TGGGTGCAGCACACCAACATGGCACATGTATACATATGTAGCAAACCTGCACGTTGTG CACATGTACCCTAGAACTTAAAGTATAATTAAAAAAAAAAAGAAAACAGAAGCTATTT ATAAAGAAGTTATTTGCTGAAATAAATGTGATCTTTCCCATTAAAAAAATAAAGAAAT TTTGGGGTAAAAAAACACAATATATTGTATTCTTGAAAAATTCTAAGAGAGTGGATGT GAAGTGTTCTCACCACAAAAGTGATAACTAATTGAGGTAATGCACATATTAATTAGAA AGATTTTGTCATTCCACAATGTATATATACTTAAAAATATGTTATACACAATAAATACA TACATTAAAAAATAAGTAAATGTA 3UTR-012 Col6a1;CCCACCCTGCACGCCGGCACCAAACCCTG 16 collagen,TCCTCCCACCCCTCCCCACTCATCACTAAA type VI, CAGAGTAAAATGTGATGCGAATTTTCCCGalpha 1 ACCAACCTGATTCGCTAGATTTTTTTTAAG GAAAAGCTTGGAAAGCCAGGACACAACGCTGCTGCCTGCTTTGTGCAGGGTCCTCCGG GGCTCAGCCCTGAGTTGGCATCACCTGCGCAGGGCCCTCTGGGGCTCAGCCCTGAGCT AGTGTCACCTGCACAGGGCCCTCTGAGGCTCAGCCCTGAGCTGGCGTCACCTGTGCAG GGCCCTCTGGGGCTCAGCCCTGAGCTGGCCTCACCTGGGTTCCCCACCCCGGGCTCTCC TGCCCTGCCCTCCTGCCCGCCCTCCCTCCTGCCTGCGCAGCTCCTTCCCTAGGCACCTCT GTGCTGCATCCCACCAGCCTGAGCAAGACGCCCTCTCGGGGCCTGTGCCGCACTAGCCT CCCTCTCCTCTGTCCCCATAGCTGGTTTTTCCCACCAATCCTCACCTAACAGTTACTTTA CAATTAAACTCAAAGCAAGCTCTTCTCCTCAGCTTGGGGCAGCCATTGGCCTCTGTCTCG TTTTGGGAAACCAAGGTCAGGAGGCCGTTGCAGACATAAATCTCGGCGACTCGGCCCC GTCTCCTGAGGGTCCTGCTGGTGACCGGCCTGGACCTTGGCCCTACAGCCCTGGAGGC CGCTGCTGACCAGCACTGACCCCGACCTCAGAGAGTACTCGCAGGGGCGCTGGCTGCA CTCAAGACCCTCGAGATTAACGGTGCTAACCCCGTCTGCTCCTCCCTCCCGCAGAGACT GGGGCCTGGACTGGACATGAGAGCCCCTTGGTGCCACAGAGGGCTGTGTCTTACTAGA AACAACGCAAACCTCTCCTTCCTCAGAATAGTGATGTGTTCGACGTTTTATCAAAGGCC CCCTTTCTATGTTCATGTTAGTTTTGCTCCTTCTGTGTTTTTTTCTGAACCATATCCATGTT GCTGACTTTTCCAAATAAAGGTTTTCACTC CTCTC3UTR-013 Calr; AGAGGCCTGCCTCCAGGGCTGGACTGAGG 17 calreticulinCCTGAGCGCTCCTGCCGCAGAGCTGGCCG CGCCAAATAATGTCTCTGTGAGACTCGAGAACTTTCATTTTTTTCCAGGCTGGTTCGGA TTTGGGGTGGATTTTGGTTTTGTTCCCCTCCTCCACTCTCCCCCACCCCCTCCCCGCCCT TTTTTTTTTTTTTTTTTAAACTGGTATTTTATCTTTGATTCTCCTTCAGCCCTCACCCCTG GTTCTCATCTTTCTTGATCAACATCTTTTCTTGCCTCTGTCCCCTTCTCTCATCTCTTAGCT CCCCTCCAACCTGGGGGGCAGTGGTGTGGAGAAGCCACAGGCCTGAGATTTCATCTGC TCTCCTTCCTGGAGCCCAGAGGAGGGCAGCAGAAGGGGGTGGTGTCTCCAACCCCCCA GCACTGAGGAAGAACGGGGCTCTTCTCATTTCACCCCTCCCTTTCTCCCCTGCCCCCAG GACTGGGCCACTTCTGGGTGGGGCAGTGGGTCCCAGATTGGCTCACACTGAGAATGTA AGAACTACAAACAAAATTTCTATTAAATTAAATTTTGTGTCTCC 3UTR-014 Col1a1; CTCCCTCCATCCCAACCTGGCTCCCTCCCA 18collagen, CCCAACCAACTTTCCCCCCAACCCGGAAA type I,CAGACAAGCAACCCAAACTGAACCCCCTC alpha 1 AAAAGCCAAAAAATGGGAGACAATTTCACATGGACTTTGGAAAATATTTTTTTCCTTTG CATTCATCTCTCAAACTTAGTTTTTATCTTTGACCAACCGAACATGACCAAAAACCAAA AGTGCATTCAACCTTACCAAAAAAAAAAAAAAAAAAAGAATAAATAAATAACTTTTTA AAAAAGGAAGCTTGGTCCACTTGCTTGAAGACCCATGCGGGGGTAAGTCCCTTTCTGC CCGTTGGGCTTATGAAACCCCAATGCTGCCCTTTCTGCTCCTTTCTCCACACCCCCCTTG GGGCCTCCCCTCCACTCCTTCCCAAATCTGTCTCCCCAGAAGACACAGGAAACAATGTA TTGTCTGCCCAGCAATCAAAGGCAATGCTCAAACACCCAAGTGGCCCCCACCCTCAGC CCGCTCCTGCCCGCCCAGCACCCCCAGGCCCTGGGGGACCTGGGGTTCTCAGACTGCC AAAGAAGCCTTGCCATCTGGCGCTCCCATGGCTCTTGCAACATCTCCCCTTCGTTTTTG AGGGGGTCATGCCGGGGGAGCCACCAGCCCCTCACTGGGTTCGGAGGAGAGTCAGGAA GGGCCACGACAAAGCAGAAACATCGGATTTGGGGAACGCGTGTCAATCCCTTGTGCCG CAGGGCTGGGCGGGAGAGACTGTTCTGTTCCTTGTGTAACTGTGTTGCTGAAAGACTAC CTCGTTCTTGTCTTGATGTGTCACCGGGGCAACTGCCTGGGGGCGGGGATGGGGGCAG GGTGGAAGCGGCTCCCCATTTTATACCAAAGGTGCTACATCTATGTGATGGGTGGGGT GGGGAGGGAATCACTGGTGCTATAGAAATTGAGATGCCCCCCCAGGCCAGCAAATGTT CCTTTTTGTTCAAAGTCTATTTTTATTCCTTGATATTTTTCTTTTTTTTTTTTTTTTTTTGTG GATGGGGACTTGTGAATTTTTCTAAAGGTGCTATTTAACATGGGAGGAGAGCGTGTGC GGCTCCAGCCCAGCCCGCTGCTCACTTTCCACCCTCTCTCCACCTGCCTCTGGCTTCTCA GGCCTCTGCTCTCCGACCTCTCTCCTCTGAAACCCTCCTCCACAGCTGCAGCCCATCCTC CCGGCTCCCTCCTAGTCTGTCCTGCGTCCTCTGTCCCCGGGTTTCAGAGACAACTTCCCA AAGCACAAAGCAGTTTTTCCCCCTAGGGGTGGGAGGAAGCAAAAGACTCTGTACCTAT TTTGTATGTGTATAATAATTTGAGATGTTTTTAATTATTTTGATTGCTGGAATAAAGCAT GTGGAAATGACCCAAACATAATCCGCAGTGGCCTCCTAATTTCCTTCTTTGGAGTTGGG GGAGGGGTAGACATGGGGAAGGGGCTTTGGGGTGATGGGCTTGCCTTCCATTCCTGCCC TTTCCCTCCCCACTATTCTCTTCTAGATCCCTCCATAACCCCACTCCCCTTTCTCTCACCC TTCTTATACCGCAAACCTTTCTACTTCCTCTTTCATTTTCTATTCTTGCAATTTCCTTGCA CCTTTTCCAAATCCTCTTCTCCCCTGCAATACCATACAGGCAATCCACGTGCACAACAC ACACACACACTCTTCACATCTGGGGTTGTCCAAACCTCATACCCACTCCCCTTCAAGCCC ATCCACTCTCCACCCCCTGGATGCCCTGCACTTGGTGGCGGTGGGATGCTCATGGATAC TGGGAGGGTGAGGGGAGTGGAACCCGTGAGGAGGACCTGGGGGCCTCTCCTTGAACT GACATGAAGGGTCATCTGGCCTCTGCTCCCTTCTCACCCACGCTGACCTCCTGCCGAAG GAGCAACGCAACAGGAGAGGGGTCTGCTGAGCCTGGCGAGGGTCTGGGAGGGACCAGG AGGAAGGCGTGCTCCCTGCTCGCTGTCCTGGCCCTGGGGGAGTGAGGGAGACAGACA CCTGGGAGAGCTGTGGGGAAGGCACTCGCACCGTGCTCTTGGGAAGGAAGGAGACCTG GCCCTGCTCACCACGGACTGGGTGCCTCGACCTCCTGAATCCCCAGAACACAACCCCC CTGGGCTGGGGTGGTCTGGGGAACCATCGTGCCCCCGCCTCCCGCCTACTCCTTTTTAA GCTT 3UTR-015 Plod1;TTGGCCAGGCCTGACCCTCTTGGACCTTTC 19 procollagen-TTCTTTGCCGACAACCACTGCCCAGCAGCC lysine, TCTGGGACCTCGGGGTCCCAGGGAACCCA 2-GTCCAGCCTCCTGGCTGTTGACTTCCCATT oxoglutarateGCTCTTGGAGCCACCAATCAAAGAGATTC 5- AAAGAGATTCCTGCAGGCCAGAGGCGGAAdioxygenase 1 CACACCTTTATGGCTGGGGCTCTCCGTGGTGTTCTGGACCCAGCCCCTGGAGACACCAT TCACTTTTACTGCTTTGTAGTGACTCGTGCTCTCCAACCTGTCTTCCTGAAAAACCAAG GCCCCCTTCCCCCACCTCTTCCATGGGGTGAGACTTGAGCAGAACAGGGGCTTCCCCAA GTTGCCCAGAAAGACTGTCTGGGTGAGAAGCCATGGCCAGAGCTTCTCCCAGGCACAG GTGTTGCACCAGGGACTTCTGCTTCAAGTTTTGGGGTAAAGACACCTGGATCAGACTCC AAGGGCTGCCCTGAGTCTGGGACTTCTGCCTCCATGGCTGGTCATGAGAGCAAACCGT AGTCCCCTGGAGACAGCGACTCCAGAGAACCTCTTGGGAGACAGAAGAGGCATCTGTG CACAGCTCGATCTTCTACTTGCCTGTGGGGAGGGGAGTGACAGGTCCACACACCACACT GGGTCACCCTGTCCTGGATGCCTCTGAAGAGAGGGACAGACCGTCAGAAACTGGAGA GTTTCTATTAAAGGTCATTTAAACCA 3UTR-016 Nucb1;TCCTCCGGGACCCCAGCCCTCAGGATTCCT 20 nucleobindin 1GATGCTCCAAGGCGACTGATGGGCGCTGG ATGAAGTGGCACAGTCAGCTTCCCTGGGGGCTGGTGTCATGTTGGGCTCCTGGGGCGG GGGCACGGCCTGGCATTTCACGCATTGCTGCCACCCCAGGTCCACCTGTCTCCACTTTC ACAGCCTCCAAGTCTGTGGCTCTTCCCTTCTGTCCTCCGAGGGGCTTGCCTTCTCTCGTG TCCAGTGAGGTGCTCAGTGATCGGCTTAACTTAGAGAAGCCCGCCCCCTCCCCTTCTCC GTCTGTCCCAAGAGGGTCTGCTCTGAGCCTGCGTTCCTAGGTGGCTCGGCCTCAGCTGCC TGGGTTGTGGCCGCCCTAGCATCCTGTATGCCCACAGCTACTGGAATCCCCGCTGCTGCT CCGGGCCAAGCTTCTGGTTGATTAATGAGGGCATGGGGTGGTCCCTCAAGACCTTCCC CTACCTTTTGTGGAACCAGTGATGCCTCAAAGACAGTGTCCCCTCCACAGCTGGGTGCC AGGGGCAGGGGATCCTCAGTATAGCCGGTGAACCCTGATACCAGGAGCCTGGGCCTCC CTGAACCCCTGGCTTCCAGCCATCTCATCGCCAGCCTCCTCCTGGACCTCTTGGCCCCCA GCCCCTTCCCCACACAGCCCCAGAAGGGTCCCAGAGCTGACCCCACTCCAGGACCTAG GCCCAGCCCCTCAGCCTCATCTGGAGCCCCTGAAGACCAGTCCCACCCACCTTTCTGGC CTCATCTGACACTGCTCCGCATCCTGCTGTGTGTCCTGTTCCATGTTCCGGTTCCATCCA AATACACTTTCTGGAACAAA 3UTR-017 α-globinGCTGGAGCCTCGGTGGCCATGCTTCTTGCC 21 CCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGA ATAAAGTCTGAGTGGGCGGC

It should be understood that those listed in the previous tables areexamples and that any UTR from any gene may be incorporated into therespective first or second flanking region of the primary construct.Furthermore, multiple wild-type UTRs of any known gene may be utilized.It is also within the scope of the present invention to provideartificial UTRs which are not variants of wild type genes. These UTRs orportions thereof may be placed in the same orientation as in thetranscript from which they were selected or may be altered inorientation or location. Hence a 5′ or 3′ UTR may be inverted,shortened, lengthened, made chimeric with one or more other 5′ UTRs or3′ UTRs. As used herein, the term “altered” as it relates to a UTRsequence, means that the UTR has been changed in some way in relation toa reference sequence. For example, a 3′ or 5′ UTR may be alteredrelative to a wild type or native UTR by the change in orientation orlocation as taught above or may be altered by the inclusion ofadditional nucleotides, deletion of nucleotides, swapping ortransposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In one embodiment, a double, triple or quadruple UTR such as a 5′ or 3′UTR may be used. As used herein, a “double” UTR is one in which twocopies of the same UTR are encoded either in series or substantially inseries. For example, a double beta-globin 3′ UTR may be used asdescribed in US Patent publication 20100129877, the contents of whichare incorporated herein by reference in its entirety.

It is also within the scope of the present invention to have patternedUTRs. As used herein “patterned UTRs” are those UTRs which reflect arepeating or alternating pattern, such as ABABAB or AABBAABBAABB orABCABCABC or variants thereof repeated once, twice, or more than 3times. In these patterns, each letter, A, B, or C represent a differentUTR at the nucleotide level.

In one embodiment, flanking regions are selected from a family oftranscripts whose proteins share a common function, structure, featureof property. For example, polypeptides of interest may belong to afamily of proteins which are expressed in a particular cell, tissue orat some time during development. The UTRs from any of these genes may beswapped for any other UTR of the same or different family of proteins tocreate a new chimeric primary transcript. As used herein, a “family ofproteins” is used in the broadest sense to refer to a group of two ormore polypeptides of interest which share at least one function,structure, feature, localization, origin, or expression pattern.

After optimization (if desired), the primary construct components arereconstituted and transformed into a vector such as, but not limited to,plasmids, viruses, cosmids, and artificial chromosomes. For example, theoptimized construct may be reconstituted and transformed into chemicallycompetent E. coli, yeast, neurospora, maize, drosophila, etc. where highcopy plasmid-like or chromosome structures occur by methods describedherein.

The untranslated region may also include translation enhancer elements(TEE). As a non-limiting example, the TEE may include those described inUS Application No. 20090226470, herein incorporated by reference in itsentirety, and those known in the art.

Stop Codons

In one embodiment, the primary constructs of the present invention mayinclude at least two stop codons before the 3′ untranslated region(UTR). The stop codon may be selected from TGA, TAA and TAG. In oneembodiment, the primary constructs of the present invention include thestop codon TGA and one additional stop codon. In a further embodimentthe addition stop codon may be TAA. In another embodiment, the primaryconstructs of the present invention include three stop codons.

Vector Amplification

The vector containing the primary construct is then amplified and theplasmid isolated and purified using methods known in the art such as,but not limited to, a maxi prep using the Invitrogen PURELINK™ HiPureMaxiprep Kit (Carlsbad, Calif.).

Plasmid Linearization

The plasmid may then be linearized using methods known in the art suchas, but not limited to, the use of restriction enzymes and buffers. Thelinearization reaction may be purified using methods including, forexample Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.), andHPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phaseHPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) andInvitrogen's standard PURELINK™ PCR Kit (Carlsbad, Calif.). Thepurification method may be modified depending on the size of thelinearization reaction which was conducted. The linearized plasmid isthen used to generate cDNA for in vitro transcription (IVT) reactions.

cDNA Template Synthesis

A cDNA template may be synthesized by having a linearized plasmidundergo polymerase chain reaction (PCR). Table 4 is a listing of primersand probes that may be usefully in the PCR reactions of the presentinvention. It should be understood that the listing is not exhaustiveand that primer-probe design for any amplification is within the skillof those in the art. Probes may also contain chemically modified basesto increase base-pairing fidelity to the target molecule andbase-pairing strength. Such modifications may include 5-methyl-Cytidine,2,6-di-amino-purine, 2′-fluoro, phosphoro-thioate, or locked nucleicacids.

TABLE 4 Primers and Probes Primer/ SEQ Probe Hybridization ID IdentifierSequence (5′-3′) target NO. UFP TTGGACCCTCGTACA cDNA Template 22GAAGCTAATACG URP T_(x160)CTTCCTACTCAGG cDNA Template 23 CTTTATTCAAAGACCAGBA1 CCTTGACCTTCTGGAACT Acid 24 TC glucocerebrosidase GBA2CCAAGCACTGAAACGGAT Acid 25 AT glucocerebrosidase LUC1 GATGAAAAGTGCTCCAAGLuciferase 26 GA LUC2 AACCGTGATGAAAAGGTA Luciferase 27 CC LUC3TCATGCAGATTGGAAAGG Luciferase 28 TC GCSF1 CTTCTTGGACTGTCCAGA G-CSF 29 GGGCSF2 GCAGTCCCTGATACAAGA G-CSF 30 AC GCSF3 GATTGAAGGTGGCTCGCT G-CSF 31AC *UFP is universal forward primer; URP is universal reverse primer.

In one embodiment, the cDNA may be submitted for sequencing analysisbefore undergoing transcription.

mRNA Production

The process of mRNA or mmRNA production may include, but is not limitedto, in vitro transcription, cDNA template removal and RNA clean-up, andmRNA capping and/or tailing reactions.

In Vitro Transcription

The cDNA produced in the previous step may be transcribed using an invitro transcription (IVT) system. The system typically comprises atranscription buffer, nucleotide triphosphates (NTPs), an RNaseinhibitor and a polymerase. The NTPs may be manufactured in house, maybe selected from a supplier, or may be synthesized as described herein.The NTPs may be selected from, but are not limited to, those describedherein including natural and unnatural (modified) NTPs. The polymerasemay be selected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to,polymerases able to incorporate modified nucleic acids.

RNA Polymerases

Any number of RNA polymerases or variants may be used in the design ofthe primary constructs of the present invention.

RNA polymerases may be modified by inserting or deleting amino acids ofthe RNA polymerase sequence. As a non-limiting example, the RNApolymerase may be modified to exhibit an increased ability toincorporate a 2′-modified nucleotide triphosphate compared to anunmodified RNA polymerase (see International Publication WO2008078180and U.S. Pat. No. 8,101,385; herein incorporated by reference in theirentireties).

Variants may be obtained by evolving an RNA polymerase, optimizing theRNA polymerase amino acid and/or nucleic acid sequence and/or by usingother methods known in the art. As a non-limiting example, T7 RNApolymerase variants may be evolved using the continuous directedevolution system set out by Esvelt et al. (Nature (2011)472(7344):499-503; herein incorporated by reference in its entirety)where clones of T7 RNA polymerase may encode at least one mutation suchas, but not limited to, lysine at position 93 substituted for threonine(K93T), 14M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T,N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A,Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P,A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A,H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E,N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limitingexample, T7 RNA polymerase variants may encode at least mutation asdescribed in U.S. Pub. Nos. 20100120024 and 20070117112; hereinincorporated by reference in their entireties. Variants of RNApolymerase may also include, but are not limited to, substitutionalvariants, conservative amino acid substitution, insertional variants,deletional variants and/or covalent derivatives.

In one embodiment, the primary construct may be designed to berecognized by the wild type or variant RNA polymerases. In doing so, theprimary construct may be modified to contain sites or regions ofsequence changes from the wild type or parent primary construct.

In one embodiment, the primary construct may be designed to include atleast one substitution and/or insertion upstream of an RNA polymerasebinding or recognition site, downstream of the RNA polymerase binding orrecognition site, upstream of the TATA box sequence, downstream of theTATA box sequence of the primary construct but upstream of the codingregion of the primary construct, within the 5′UTR, before the 5′UTRand/or after the 5′UTR.

In one embodiment, the 5′UTR of the primary construct may be replaced bythe insertion of at least one region and/or string of nucleotides of thesame base. The region and/or string of nucleotides may include, but isnot limited to, at least 3, at least 4, at least 5, at least 6, at least7 or at least 8 nucleotides and the nucleotides may be natural and/orunnatural. As a non-limiting example, the group of nucleotides mayinclude 5-8 adenine, cytosine, thymine, a string of any of the othernucleotides disclosed herein and/or combinations thereof.

In one embodiment, the 5′UTR of the primary construct may be replaced bythe insertion of at least two regions and/or strings of nucleotides oftwo different bases such as, but not limited to, adenine, cytosine,thymine, any of the other nucleotides disclosed herein and/orcombinations thereof. For example, the 5′UTR may be replaced byinserting 5-8 adenine bases followed by the insertion of 5-8 cytosinebases. In another example, the 5′UTR may be replaced by inserting 5-8cytosine bases followed by the insertion of 5-8 adenine bases.

In one embodiment, the primary construct may include at least onesubstitution and/or insertion downstream of the transcription start sitewhich may be recognized by an RNA polymerase. As a non-limiting example,at least one substitution and/or insertion may occur downstream thetranscription start site by substituting at least one nucleic acid inthe region just downstream of the transcription start site (such as, butnot limited to, +1 to +6). Changes to region of nucleotides justdownstream of the transcription start site may affect initiation rates,increase apparent nucleotide triphosphate (NTP) reaction constantvalues, and increase the dissociation of short transcripts from thetranscription complex curing initial transcription (Brieba et al,Biochemistry (2002) 41: 5144-5149; herein incorporated by reference inits entirety). The modification, substitution and/or insertion of atleast one nucleic acid may cause a silent mutation of the nucleic acidsequence or may cause a mutation in the amino acid sequence.

In one embodiment, the primary construct may include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12 or at least 13 guanine bases downstream of the transcription startsite.

In one embodiment, the primary construct may include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5 or at least 6guanine bases in the region just downstream of the transcription startsite. As a non-limiting example, if the nucleotides in the region areGGGAGA the guanine bases may be substituted by at least 1, at least 2,at least 3 or at least 4 adenine nucleotides. In another non-limitingexample, if the nucleotides in the region are GGGAGA the guanine basesmay be substituted by at least 1, at least 2, at least 3 or at least 4cytosine bases. In another non-limiting example, if the nucleotides inthe region are GGGAGA the guanine bases may be substituted by at least1, at least 2, at least 3 or at least 4 thymine, and/or any of thenucleotides described herein.

In one embodiment, the primary construct may include at least onesubstitution and/or insertion upstream of the start codon. For thepurpose of clarity, one of skill in the art would appreciate that thestart codon is the first codon of the protein coding region whereas thetranscription start site is the site where transcription begins. Theprimary construct may include, but is not limited to, at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7 orat least 8 substitutions and/or insertions of nucleotide bases. Thenucleotide bases may be inserted or substituted at 1, at least 1, atleast 2, at least 3, at least 4 or at least 5 locations upstream of thestart codon. The nucleotides inserted and/or substituted may be the samebase (e.g., all A or all C or all T or all G), two different bases(e.g., A and C, A and T, or C and T), three different bases (e.g., A, Cand T or A, C and T) or at least four different bases. As a non-limitingexample, the guanine base upstream of the coding region in the primaryconstruct may be substituted with adenine, cytosine, thymine, or any ofthe nucleotides described herein. In another non-limiting example thesubstitution of guanine bases in the primary construct may be designedso as to leave one guanine base in the region downstream of thetranscription start site and before the start codon (see Esvelt et al.Nature (2011) 472(7344):499-503; herein incorporated by reference in itsentirety). As a non-limiting example, at least 5 nucleotides may beinserted at 1 location downstream of the transcription start site butupstream of the start codon and the at least 5 nucleotides may be thesame base type.

cDNA Template Removal and Clean-Up

The cDNA template may be removed using methods known in the art such as,but not limited to, treatment with Deoxyribonuclease I (DNase I). RNAclean-up may also include a purification method such as, but not limitedto, AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, Mass.),HPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phaseHPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).

Capping and/or Tailing Reactions

The primary construct or mmRNA may also undergo capping and/or tailingreactions. A capping reaction may be performed by methods known in theart to add a 5′ cap to the 5′ end of the primary construct. Methods forcapping include, but are not limited to, using a Vaccinia Capping enzyme(New England Biolabs, Ipswich, Mass.).

A poly-A tailing reaction may be performed by methods known in the art,such as, but not limited to, 2′ O-methyltransferase and by methods asdescribed herein. If the primary construct generated from cDNA does notinclude a poly-T, it may be beneficial to perform the poly-A-tailingreaction before the primary construct is cleaned.

mRNA Purification

Primary construct or mmRNA purification may include, but is not limitedto, mRNA or mmRNA clean-up, quality assurance and quality control. mRNAor mmRNA clean-up may be performed by methods known in the arts such as,but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers,Mass.), poly-T beads, LNA™ oligo-T capture probes (EXIQON® Inc, Vedbaek,Denmark) or HPLC based purification methods such as, but not limited to,strong anion exchange HPLC, weak anion exchange HPLC, reverse phaseHPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term“purified” when used in relation to a polynucleotide such as a “purifiedmRNA or mmRNA” refers to one that is separated from at least onecontaminant. As used herein, a “contaminant” is any substance whichmakes another unfit, impure or inferior. Thus, a purified polynucleotide(e.g., DNA and RNA) is present in a form or setting different from thatin which it is found in nature, or a form or setting different from thatwhich existed prior to subjecting it to a treatment or purificationmethod.

A quality assurance and/or quality control check may be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC.

In another embodiment, the mRNA or mmRNA may be sequenced by methodsincluding, but not limited to reverse-transcriptase-PCR.

In one embodiment, the mRNA or mmRNA may be quantified using methodssuch as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).A non-limiting example of a UV/Vis spectrometer is a NANODROP®spectrometer (ThermoFisher, Waltham, Mass.). The quantified mRNA ormmRNA may be analyzed in order to determine if the mRNA or mmRNA may beof proper size, check that no degradation of the mRNA or mmRNA hasoccurred. Degradation of the mRNA and/or mmRNA may be checked by methodssuch as, but not limited to, agarose gel electrophoresis, HPLC basedpurification methods such as, but not limited to, strong anion exchangeHPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), andhydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-massspectrometry (LCMS), capillary electrophoresis (CE) and capillary gelelectrophoresis (CGE).

Signal Sequences

The primary constructs or mmRNA may also encode additional featureswhich facilitate trafficking of the polypeptides to therapeuticallyrelevant sites. One such feature which aids in protein trafficking isthe signal sequence. As used herein, a “signal sequence” or “signalpeptide” is a polynucleotide or polypeptide, respectively, which is fromabout 9 to 200 nucleotides β-60 amino acids) in length which isincorporated at the 5′ (or N-terminus) of the coding region orpolypeptide encoded, respectively. Addition of these sequences result intrafficking of the encoded polypeptide to the endoplasmic reticulumthrough one or more secretory pathways. Some signal peptides are cleavedfrom the protein by signal peptidase after the proteins are transported.

Table 5 is a representative listing of protein signal sequences whichmay be incorporated for encoding by the polynucleotides, primaryconstructs or mmRNA of the invention.

TABLE 5 Signal Sequences SEQ NUCLEOTIDE SEQUENCE ID ENCODED SEQ ID IDDescription (5′-3′) NO. PEPTIDE NO. SS-001 α-1- ATGATGCCATCCTCAGTCTCA 32MMPSSVSWGI 94 antitrypsin TGGGGTATTTTGCTCTTGGCG LLAGLCCLVPGGTCTGTGCTGTCTCGTGCCG VSLA GTGTCGCTCGCA SS-002 G-CSFATGGCCGGACCGGCGACTCAG 33 MAGPATQSPM 95 TCGCCCATGAAACTCATGGCC KLMALQLLLWCTGCAGTTGTTGCTTTGGCAC HSALWTVQEA TCAGCCCTCTGGACCGTCCAA GAGGCG SS-003Factor IX ATGCAGAGAGTGAACATGATT 34 MQRVNMIMAE 96 ATGGCCGAGTCCCCATCGCTCSPSLITICLLGY ATCACAATCTGCCTGCTTGGT LLSAECTVFLD ACCTGCTTTCCGCCGAATGCAHENANKILNRP CTGTCTTTCTGGATCACGAGA KR ATGCGAATAAGATCTTGAACC GACCCAAACGGSS-004 Prolactin ATGAAAGGATCATTGCTGTTG 35 MKGSLLLLLV 97CTCCTCGTGTCGAACCTTCTG SNLLLCQSVAP CTTTGCCAGTCCGTAGCCCCC SS-005 AlbuminATGAAATGGGTGACGTTCATC 36 MKWVTFISLLF 98 TCACTGTTGTTTTTGTTCTCGT LFSSAYSRGCCGCCTACTCCAGGGGAGTAT VFRR TCCGCCGA SS-006 HMMSP38 ATGTGGTGGCGGCTCTGGTGG37 MWWRLWWLL 99 CTGCTCCTGTTGCTCCTCTTGC LLLLLLPMWA TGTGGCCCATGGTGTGGGCAMLS- ornithine TGCTCTTTAACCTCCGCATCCT 38 MLFNLRILLNN 100 001carbamoyltransferase GTTGAATAACGCTGCGTTCCG AAFRNGHNFMAAATGGGCATAACTTCATGGT VRNFRCGQPLQ ACGCAACTTCAGATGCGGCCA GCCACTCCAG MLS-Cytochrome ATGTCCGTCTTGACACCCCTG 39 MSVLTPLLLRG 101 002 C OxidaseCTCTTGAGAGGGCTGACGGGG LTGSARRLPVP subunit 8A TCCGCTAGACGCCTGCCGGTARAKIHSL CCGCGAGCGAAGATCCACTCC CTG MLS- Cytochrome ATGAGCGTGCTCACTCCGTTG40 MSVLTPLLLRG 102 003 C Oxidase CTTCTTCGAGGGCTTACGGGA LTGSARRLPVPsubunit 8A TCGGCTCGGAGGTTGCCCGTC RAKIHSL CCGAGAGCGAAGATCCATTCG TTGSS-007 Type III, TGACAAAAATAACTTTATCTC 41 MVTKITLSPQN 103 bacterialCCCAGAATTTTAGAATCCAAA FRIQKQETTLL AACAGGAAACCACACTACTA KEKSTEKNSLAAAAGAAAAATCAACCGAGAA KSILAVKNHFI AAATTCTTTAGCAAAAAGTAT ELRSKLSERFISTCTCGCAGTAAAAATCACTTC HKNT ATCGAATTAAGGTCAAAATTA TCGGAACGTTTTATTTCGCATAAGAACACT SS-008 Viral ATGCTGAGCTTTGTGGATACC 42 MLSFVDTRTLL 104CGCACCCTGCTGCTGCTGGCG LLAVTSCLATCQ GTGACCAGCTGCCTGGCGACC TGCCAG SS-009viral ATGGGCAGCAGCCAGGCGCC 43 MGSSQAPRMG 105 GCGCATGGGCAGCGTGGGCGSVGGHGLMAL GCCATGGCCTGATGGCGCTGC LMAGLILPGILA TGATGGCGGGCCTGATTCTGCCGGGCATTCTGGCG SS-010 Viral ATGGCGGGCATTTTTTATTTTC 44 MAGIFYFLFSF 106TGTTTAGCTTTCTGTTTGGCAT LFGICD TTGCGAT SS-011 Viral ATGGAAAACCGCCTGCTGCGC45 MENRLLRVFL 107 GTGTTTCTGGTGTGGGCGGCG VWAALTMDG CTGACCATGGATGGCGCGAGCASA GCG SS-012 Viral ATGGCGCGCCAGGGCTGCTTT 46 MARQGCFGSY 108GGCAGCTATCAGGTGATTAGC QVISLFTFAIGV CTGTTTACCTTTGCGATTGGC NLCLGGTGAACCTGTGCCTGGGC SS-013 Bacillus ATGAGCCGCCTGCCGGTGCTG 47 MSRLPVLLLLQ109 CTGCTGCTGCAGCTGCTGGTG LLVRPGLQ CGCCCGGGCCTGCAG SS-014 BacillusATGAAACAGCAGAAACGCCT 48 MKQQKRLYAR 110 GTATGCGCGCCTGCTGACCCTLLTLLFALIFLL GCTGTTTGCGCTGATTTTTCTG PHSSASA CTGCCGCATAGCAGCGCGAGC GCGSS-015 Secretion ATGGCGACGCCGCTGCCTCCG 49 MATPLPPPSPR 111 signalCCCTCCCCGCGGCACCTGCGG HLRLLRLLLSG CTGCTGCGGCTGCTGCTCTCC GCCCTCGTCCTCGGCSS-016 Secretion ATGAAGGCTCCGGGTCGGCTC 50 MKAPGRLVLII 112 signalGTGCTCATCATCCTGTGCTCC LCSVVFS GTGGTCTTCTCT SS-017 SecretionATGCTTCAGCTTTGGAAACTT 51 MLQLWKLLCG 113 signal GTTCTCCTGTGCGGCGTGCTC VLTACT SS-018 Secretion ATGCTTTATCTCCAGGGTTGG 52 MLYLQGWSM 114 signalAGCATGCCTGCTGTGGCA PAVA SS-019 Secretion ATGGATAACGTGCAGCCGAA 53MDNVQPKIKH 115 signal AATAAAACATCGCCCCTTCTG RPFCFSVKGHVCTTCAGTGTGAAAGGCCACGT KMLRLDIINSL GAAGATGCTGCGGCTGGATAT VTTVFMLIVSVTATCAACTCACTGGTAACAAC LALIP AGTATTCATGCTCATCGTATC TGTGTTGGCACTGATACCASS-020 Secretion ATGCCCTGCCTAGACCAACAG 54 MPCLDQQLTV 116 signalCTCACTGTTCATGCCCTACCCT HALPCPAQPSS GCCCTGCCCAGCCCTCCTCTC LAFCQVGFLTATGGCCTTCTGCCAAGTGGGGT TCTTAACAGCA SS-021 Secretion ATGAAAACCTTGTTCAATCCA55 MKTLFNPAPAI 117 signal GCCCCTGCCATTGCTGACCTG ADLDPQFYTLSGATCCCCAGTTCTACACCCTC DVFCCNESEAE TCAGATGTGTTCTGCTGCAAT ILTGLTVGSAAGAAAGTGAGGCTGAGATTTTA DA ACTGGCCTCACGGTGGGCAGC GCTGCAGATGCT SS-022Secretion ATGAAGCCTCTCCTTGTTGTG 56 MKPLLVVFVF 118 signalTTTGTCTTTCTTTTCCTTTGGG LFLWDPVLA ATCCAGTGCTGGCA SS-023 SecretionATGTCCTGTTCCCTAAAGTTT 57 MSCSLKFTLIVI 119 signal ACTTTGATTGTAATTTTTTTTTFFTCTLSSS ACTGTTGGCTTTCATCCAGC SS-024 Secretion ATGGTTCTTACTAAACCTCTTC58 MVLTKPLQRN 120 signal AAAGAAATGGCAGCATGATG GSMMSFENVKAGCTTTGAAAATGTGAAAGAA EKSREGGPHA AAGAGCAGAGAAGGAGGGCC HTPEEELCFVVCCATGCACACACACCCGAAGA THTPQVQTTL AGAATTGTGTTTCGTGGTAAC NLFFHIFKVLTACACTACCCTCAGGTTCAGAC QPLSLLWG CACACTCAACCTGTTTTTCCATATATTCAAGGTTCTTACTCAA CCACTTTCCCTTCTGTGGGGT SS-025 SecretionATGGCCACCCCGCCATTCCGG 59 MATPPFRLIRK 121 signal CTGATAAGGAAGATGTTTTCCMFSFKVSRWM TTCAAGGTGAGCAGATGGATG GLACFRSLAAS GGGCTTGCCTGCTTCCGGTCCCTGGCGGCATCC SS-026 Secretion ATGAGCTTTTTCCAACTCCTG 60 MSFFQLLMKR 122signal ATGAAAAGGAAGGAACTCAT KELIPLVVFMT TCCCTTGGTGGTGTTCATGAC VAAGGASSTGTGGCGGCGGGTGGAGCCTC ATCT SS-027 Secretion ATGGTCTCAGCTCTGCGGGGA 61MVSALRGAPLI 123 signal GCACCCCTGATCAGGGTGCAC RVHSSPVSSPSTCAAGCCCTGTTTCTTCTCCTT VSGPAALVSCL CTGTGAGTGGACCACGGAGGC SSQSSALSTGGTGAGCTGCCTGTCATCCC AAAGCTCAGCTCTGAGC SS-028 SecretionATGATGGGGTCCCCAGTGAGT 62 MMGSPVSHLL 124 signal CATCTGCTGGCCGGCTTCTGTAGFCVWVVLG GTGTGGGTCGTCTTGGGC SS-029 Secretion ATGGCAAGCATGGCTGCCGTG 63MASMAAVLT 125 signal CTCACCTGGGCTCTGGCTCTT WALALLSAFSCTTTCAGCGTTTTCGGCCACC ATQA CAGGCA SS-030 Secretion ATGGTGCTCATGTGGACCAGT64 MVLMWTSGD 126 signal GGTGACGCCTTCAAGACGGCC AFKTAYFLLKTACTTCCTGCTGAAGGGTGCC GAPLQFSVCGL CCTCTGCAGTTCTCCGTGTGC LQVLVDLAILGGGCCTGCTGCAGGTGCTGGTG QATA GACCTGGCCATCCTGGGGCAG GCCTACGCC SS-031Secretion ATGGATTTTGTCGCTGGAGCC 65 MDFVAGAIGG 127 signalATCGGAGGCGTCTGCGGTGTT VCGVAVGYPL GCTGTGGGCTACCCCCTGGAC DTVKVRIQTEPACGGTGAAGGTCAGGATCCA LYTGIWHCVR GACGGAGCCAAAGTACACAG DTYHRERVWGGCATCTGGCACTGCGTCCGGG FYRGLSLPVCT ATACGTATCACCGAGAGCGCG VSLVSS TGTGGGGCTTCTACCGGGGCCTCTCGC TGCCCGTGTGCACGGTGTCCC TGGTATCTTCC SS-032 SecretionATGGAGAAGCCCCTCTTCCCA 66 MEKPLFPLVPL 128 signal TTAGTGCCTTTGCATTGGTTTGHWFGFGYTAL GCTTTGGCTACACAGCACTGG VVSGGIVGYV TTGTTTCTGGTGGGATCGTTGKTGSVPSLAA GCTATGTAAAAACAGGCAGC GLLFGSLA GTGCCGTCCCTGGCTGCAGGGCTGCTCTTCGGCAGTCTAGCC SS-033 Secretion ATGGGTCTGCTCCTTCCCCTG 67MGLLLPLALCI 129 signal GCACTCTGCATCCTAGTCCTG LVLC TGC SS-034 SecretionATGGGGATCCAGACGAGCCCC 68 MGIQTSPVLLA 130 signal GTCCTGCTGGCCTCCCTGGGGSLGVGLVTLL GTGGGGCTGGTCACTCTGCTC GLAVG GGCCTGGCTGTGGGC SS-035 SecretionATGTCGGACCTGCTACTACTG 69 MSDLLLLGLIG 131 signal GGCCTGATTGGGGGCCTGACTGLTLLLLLTLL CTCTTACTGCTGCTGACGCTG AFA CTAGCCTTTGCC SS-036 SecretionATGGAGACTGTGGTGATTGTT 70 METVVIVAIGV 132 signal GCCATAGGTGTGCTGGCCACCLATIFLASFAA ATGTTTCTGGCTTCGTTTGCAG LVLVCRQ CCTTGGTGCTGGTTTGCAGGC AGSS-037 Secretion ATGCGCGGCTCTGTGGAGTGC 71 MAGSVECTWG 133 signalACCTGGGGTTGGGGGCACTGT WGHCAPSPLL GCCCCCAGCCCCCTGCTCCTT LWTLLLFAAPFTGGACTCTACTTCTGTTTGCA GLLG GCCCCATTTGGCCTGCTGGGG SS-038 SecretionATGATGCCGTCCCGTACCAAC 72 MMPSRTNLAT 134 signal CTGGCTACTGGAATCCCCAGTGIPSSKVKYSR AGTAAAGTGAAATATTCAAGG LSSTDDGYIDL CTCTCCAGCACAGACGATGGCQFKKTPPKIPY TACATTGACCTTCAGTTTAAG KAIALATVLFL AAAACCCCTCCTAAGATCCCT IGATATAAGGCCATCGCACTTGCC ACTGTGCTGTTTTTGATTGGC GCC SS-039 SecretionATGGCCCTGCCCCAGATGTGT 73 MALPQMCDGS 135 signal GACGGGAGCCACTTGGCCTCCHLASTLRYCM ACCCTCCGCTATTGCATGACA TVSGTVVLVA GTCAGCGGCACAGTGGTTCTG GTLCFAGTGGCCGGGACGCTCTGCTTC GCT SS-041 Vrg-6 TGAAAAAGTGGTTCGTTGCTG 74MKKWFVAAGI 136 CCGGCATCGGCGCTGCCGGAC GAGLLMLSSAA TCATGCTCTCCAGCGCCGCCASS-042 PhoA ATGAAACAGAGCACCATTGCG 75 MKQSTIALALL 137CTGGCGCTGCTGCCGCTGCTG PLLFTPVTKA TTTACCCCGGTGACCAAAGCG SS-043 OmpAATGAAAAAAACCGCGATTGC 76 MKKTAIAIAV 138 GATTGCGGTGGCGCTGGCGGG ALAGFATVAQACTTTGCGACCGTGGCGCAGGCG SS-044 STI ATGAAAAAACTGATGCTGGCG 77 MKKLMLAIFFS139 ATTTTTTTTAGCGTGCTGAGCT VLSFPSFSQS TTCCGAGCTTTAGCCAGAGC SS-045 STIIATGAAAAAAAACATTGCGTTT 78 MKKNIAFLLAS 140 CTGCTGGCGAGCATGTTTGTGMFVFSIATNAYA TTTAGCATTGCGACCAACGCG TATGCG SS-046 AmylaseATGTTTGCGAAACGCTTTAAA 79 MFAKRFKTSL 141 ACCAGCCTGCTGCCGCTGTTTLPLFAGFLLLF GCGGGCTTTCTGCTGCTGTTTC HLVLAGPAAAS ATCTGGTGCTGGCGGGCCCGGCGGCGGCGAGC SS-047 Alpha ATGCGCTTTCCGAGCATTTTT 80 MRFPSIFTAVL 142 FactorACCGCGGTGCTGTTTGCGGCG FAASSALA AGCAGCGCGCTGGCG SS-048 AlphaATGCGCTTTCCGAGCATTTTT 81 MRFPSIFTTVL 143 Factor ACCACCGTGCTGTTTGCGGCGFAASSALA AGCAGCGCGCTGGCG SS-049 Alpha ATGCGCTTTCCGAGCATTTTT 82MRFPSIFTSVLF 144 Factor ACCAGCGTGCTGTTTGCGGCG AASSALA AGCAGCGCGCTGGCGSS-050 Alpha ATGCGCTTTCCGAGCATTTTT 83 MRFPSIFTHVL 145 FactorACCCATGTGCTGTTTGCGGCG FAASSALA AGCAGCGCGCTGGCG SS-051 AlphaATGCGCTTTCCGAGCATTTTT 84 MRFPSIFTIVLF 146 Factor ACCATTGTGCTGTTTGCGGCGAASSALA AGCAGCGCGCTGGCG SS-052 Alpha ATGCGCTTTCCGAGCATTTTT 85MRFPSIFTFVLF 147 Factor ACCTTTGTGCTGTTTGCGGCG AASSALA AGCAGCGCGCTGGCGSS-053 Alpha ATGCGCTTTCCGAGCATTTTT 86 MRFPSIFTEVL 148 FactorACCGAAGTGCTGTTTGCGGCG FAASSALA AGCAGCGCGCTGGCG SS-054 AlphaATGCGCTTTCCGAGCATTTTT 87 MRFPSIFTGVL 149 Factor ACCGGCGTGCTGTTTGCGGCGFAASSALA AGCAGCGCGCTGGCG SS-055 Endoglucanase V ATGCGTTCCTCCCCCCTCCTCC88 MRSSPLLRSAV 150 GCTCCGCCGTTGTGGCCGCCC VAALPVLALA TGCCGGTGTTGGCCCTTGCCSS-056 Secretion ATGGGCGCGGCGGCCGTGCGC 89 MGAAAVRWH 151 signalTGGCACTTGTGCGTGCTGCTG LCVLLALGTR GCCCTGGGCACACGCGGGCG GRL GCTG SS-057Fungal ATGAGGAGCTCCCTTGTGCTG 90 MRSSLVLFFVS 152 TTCTTTGTCTCTGCGTGGACGAWTALA GCCTTGGCCAG SS-058 Fibronectin ATGCTCAGGGGTCCGGGACCC 91MLRGPGPGRL 153 GGGCGGCTGCTGCTGCTAGCA LLLAVLCLGTS GTCCTGTGCCTGGGGACATCGVRCTETGKSKR GTGCGCTGCACCGAAACCGGG AAGAGCAAGAGG SS-059 FibronectinATGCTTAGGGGTCCGGGGCCC 92 MLRGPGPGLL 154 GGGCTGCTGCTGCTGGCCGTC LLAVQCLGTACAGCTGGGGACAGCGGTGCCC VPSTGA TCCACG SS-060 FibronectinATGCGCCGGGGGGCCCTGACC 93 MRRGALTGLL 155 GGGCTGCTCCTGGTCCTGTGC LVLCLSVVLRCTGAGTGTTGTGCTACGTGCA AAPSATSKKRR GCCCCCTCTGCAACAAGCAAG AAGCGCAGG

In the table, SS is secretion signal and MLS is mitochondrial leadersignal. The primary constructs or mmRNA of the present invention may bedesigned to encode any of the signal sequences of SEQ ID NOs 94-155, orfragments or variants thereof. These sequences may be included at thebeginning of the polypeptide coding region, in the middle or at theterminus or alternatively into a flanking region. Further, any of thepolynucleotide primary constructs of the present invention may alsocomprise one or more of the sequences defined by SEQ ID NOs 32-93. Thesemay be in the first region or either flanking region.

Additional signal sequences which may be utilized in the presentinvention include those taught in, for example, databases such as thosefound at http://www.signalpeptide.de/ orhttp://proline.bic.nus.edu.sg/spdb/. Those described in U.S. Pat. Nos.8,124,379; 7,413,875 and 7,385,034 are also within the scope of theinvention and the contents of each are incorporated herein by referencein their entirety.

Target Selection

According to the present invention, the primary constructs comprise atleast a first region of linked nucleosides encoding at least onepolypeptide of interest. The polypeptides of interest or “Targets” ofthe present invention are listed in Table 6. Shown in Table 6, inaddition to the name and description of the gene encoding thepolypeptide of interest are the ENSEMBL Transcript ID (ENST), theENSEMBL Protein ID (ENSP) and when available the optimized transcriptsequence ID (Optim Trans SEQ ID) or optimized open reading framesequence ID (Optim ORF SEQ ID). For any particular gene there may existone or more variants or isoforms. Where these exist, they are shown inthe table as well. It will be appreciated by those of skill in the artthat disclosed in the Table are potential flanking regions. These areencoded in each ENST transcript either to the 5′ (upstream) or 3′(downstream) of the ORF or coding region. The coding region isdefinitively and specifically disclosed by teaching the ENSP sequence.Consequently, the sequences taught flanking that encoding the proteinare considered flanking regions. It is also possible to furthercharacterize the 5′ and 3′ flanking regions by utilizing one or moreavailable databases or algorithms. Databases have annotated the featurescontained in the flanking regions of the ENST transcripts and these areavailable in the art.

TABLE 6 Targets Optim Trans Peptide Trans Target Target SEQ SEQ ID OptimORF SEQ ID No. Description ENST ID NO ENSP NO SEQ ID NO 1 4- 289004 156289004 769 1673, 2293, hydroxyphenylpyruvate 2913, 3533, dioxygenase4153, 5433-5519 2 4- 543163 157 441677 770 1674, 2294,hydroxyphenylpyruvate 2914, 3534, dioxygenase 4154 3 4- 545969 158437419 771 1675, 2295, hydroxyphenylpyruvate 2915, 3535, dioxygenase4155 4 6- 280362 159 280362 772 1676, 2296, 1609pyruvoyltetrahydropterin 2916, 3536, synthase 4156, 4773, 5103,5520-5606 5 A disintegrin and 563979 160 456186 773 1677, 2297, 1609metalloproteinase 2917, 3537, with 4157, 4774, thrombospondin 5104,5607-5693 motifs 13 isoform 1 preproprotein 6 activity-dependent 349014161 342905 774 1678, 2298, neuroprotector 2918, 3538, homeobox 4158,4775, 5105, 5694-5780 7 activity-dependent 371602 162 360662 775 1679,2299, neuroprotector 2919, 3539, homeobox 4159, 4776, 5106 8activity-dependent 396029 163 379346 776 1680, 2300, neuroprotector2920, 3540, homeobox 4160, 4777, 5107 9 activity-dependent 396032 164379349 777 1681, 2301, neuroprotector 2921, 3541, homeobox 4161, 4778,5108 10 activity-dependent 534467 165 436181 778 1682, 2302,neuroprotector 2922, 3542, homeobox 4162, 4779, 5109 11 ADAM 338351 166345120 779 1683, 2303, metallopeptidase 2923, 3543, with 4163, 4780,thrombospondin 5110 type 1 motif, 13 12 ADAM 355699 167 347927 780 1393,1684, metallopeptidase 2304, 2924, with 3544, 4164, thrombospondin 4781,5111 type 1 motif, 13 13 ADAM 356589 168 348997 781 1394, 1685,metallopeptidase 2305, 2925, with 3545, 4165, thrombospondin 4782, 5112type 1 motif, 13 14 ADAM 371910 169 360978 782 1395, 1686,metallopeptidase 2306, 2926, with 3546, 4166, thrombospondin 4783, 5113type 1 motif, 13 15 ADAM 371911 170 360979 783 1396, 1687,metallopeptidase 2307, 2927, with 3547, 4167, thrombospondin 4784, 5114type 1 motif, 13 16 ADAM 371916 171 360984 784 1397, 1688,metallopeptidase 2308, 2928, with 3548, 4168, thrombospondin 4785, 5115type 1 motif, 13 17 ADAM 371929 172 360997 785 1398, 1689, 1610metallopeptidase 2309, 2929, with 3549, 4169, thrombospondin 4786, 5116type 1 motif, 13 18 ADAM 536611 173 444504 786 1399, 1690,metallopeptidase 2310, 2930, with 3550, 4170, thrombospondin 4787, 5117type 1 motif, 13 19 AIFsh 20 Albiglutide; albiglutide 21 aldolase A563060 174 455800 787 1691, 2311, fructose- 2931, 3551, bisphosphate4171 22 aldolase A 564546 175 455917 788 1692, 2312, fructose- 2932,3552, bisphosphate 4172 23 aldolase A 564595 176 457468 789 1693, 2313,fructose- 2933, 3553, bisphosphate 4173 24 aldolase A, 338110 177 336927790 1694, 2314, fructose- 2934, 3554, bisphosphate 4174 25 aldolase A,395240 178 378661 791 1695, 2315, fructose- 2935, 3555, bisphosphate4175 26 aldolase A, 395248 179 378669 792 1696, 2316, fructose- 2936,3556, bisphosphate 4176, 5781-5867 27 aldolase A, 412304 180 400452 7931697, 2317, fructose- 2937, 3557, bisphosphate 4177 28 ameloblastin322937 181 313809 794 1698, 2318, 1611 (enamel matrix 2938, 3558,protein) 4178, 4788, 5118, 5868-5954 29 ameloblastin 449493 182 391234795 1699, 2319, 1611 (enamel matrix 2939, 3559, protein) 4179, 4789,5119 30 ameloblastin 538728 183 445605 796 1700, 2320, 1611 (enamelmatrix 2940, 3560, protein) 4180, 4790, 5120 31 amelogenin, X- 348912184 335312 797 1701, 2321, linked 2941, 3561, 4181, 4791, 5121 32amelogenin, X- 380712 185 370088 798 1702, 2322, linked 2942, 3562,4182, 4792, 5122, 5955-6041 33 amelogenin, X- 380714 186 370090 7991703, 2323, linked 2943, 3563, 4183, 4793, 5123 34 amelogenin, Y- 215479187 215479 800 1704, 2324, 1612 linked 2944, 3564, 4184 35 amelogenin,Y- 383036 188 372505 801 1705, 2325, 1612 linked 2945, 3565, 4185 36amelogenin, Y- 383037 189 372506 802 1706, 2326, 1612 linked 2946, 3566,4186 37 amylo-alpha-1,6- 294724 190 294724 803 1707, 2327, glucosidase,4- 2947, 3567, alpha- 4187 glucanotransferase 38 amylo-alpha-1,6- 361302191 354971 804 1708, 2328, glucosidase, 4- 2948, 3568, alpha- 4188glucanotransferase 39 amylo-alpha-1,6- 361522 192 354635 805 1709, 2329,glucosidase, 4- 2949, 3569, alpha- 4189 glucanotransferase 40amylo-alpha-1,6- 361915 193 355106 806 1710, 2330, glucosidase, 4- 2950,3570, alpha- 4190, 6042-6128 glucanotransferase 41 amylo-alpha-1,6-370161 194 359180 807 1711, 2331, glucosidase, 4- 2951, 3571, alpha-4191 glucanotransferase 42 amylo-alpha-1,6- 370163 195 359182 808 1712,2332, glucosidase, 4- 2952, 3572, alpha- 4192 glucanotransferase 43amylo-alpha-1,6- 370165 196 359184 809 1713, 2333, glucosidase, 4- 2953,3573, alpha- 4193 glucanotransferase 44 amyloid P 255040 197 255040 8101714, 2334, component, serum 2954, 3574, 4194, 6129-6215 45 angiotensinI 252519 198 252519 811 1715, 2335, converting enzyme 2955, 3575,(peptidyl- 4195, 6216-6302 dipeptidase A) 2 46 angiotensin I 427411 199389326 812 1716, 2336, converting enzyme 2956, 3576, (peptidyl- 4196dipeptidase A) 2 47 Anti-TNFalpha (TNFRFusion; Enbrel) 48 APOA1 Milano;813 1400, 1717, 1613 apo A- 2337, 2957, I(R173C)Milano, 3577, 4197,ETC-216 4794, 5124 49 APOA1 Paris; apo 814 1401, 1718, A-I(R151C)Paris2338, 2958, 3578, 4198, 4795, 5125 50 ApoA-a optimized 1614 mRNA 51apolipoprotein A-I 236850 200 236850 815 1402, 1719, 1615 2339, 2959,3579, 4199, 4796, 5126, 6303-5363 52 apolipoprotein A-I 359492 201352471 816 1720, 2340, 1615 2960, 3580, 4200, 4797, 5127 53apolipoprotein A-I 375320 202 364469 817 1721, 2341, 1615 2961, 3581,4201, 4798, 5128 54 apolipoprotein A-I 375323 203 364472 818 1722, 2342,1615 2962, 3582, 4202, 4799, 5129 55 apolipoprotein B 233242 204 233242819 1723, 2343, (including Ag(x) 2963, 3583, antigen) 4203 56apolipoprotein B 535079 205 439731 820 1724, 2344, (including Ag(x)2964, 3584, antigen) 4204 57 aquaporin 5 293599 206 293599 821 1725,2345, 1616 2965, 3585, 4205, 4800, 5130, 6564-6650 58 arginase, liver356962 207 349446 822 1726, 2346, 2966, 3586, 4206, 59 arginase, liver368087 208 357066 823 1727, 2347, 2967, 3587, 4207, 6651-6737 60arginase, liver 476845 209 417694 824 1728, 2348, 2968, 3588, 4208 61argininosuccinate 304874 210 307188 825 1729, 2349, lyase 2969, 3589,4209, 6738-6876 62 argininosuccinate 380839 211 370219 826 1730, 2350,lyase 2970, 3590, 4210 63 argininosuccinate 395331 212 378740 827 1731,2351, lyase 2971, 3591, 4211 64 argininosuccinate 395332 213 378741 8281732, 2352, lyase 2972, 3592, 4212 65 argininosuccinate 502022 214441778 829 1733, 2353, lyase 2973, 3593, 4213 66 argininosuccinate334909 215 361470 830 1403, 1734, synthase 1 2354, 2974, 3594, 4214 67argininosuccinate 352480 216 253004 831 1735, 2355, synthase 1 2975,3595, 4215, 6877-6963 68 argininosuccinate 372386 217 361461 832 1404,1736, synthase 1 2356, 2976, 3596, 4216 69 argininosuccinate 372393 218361469 833 1737, 2357, synthase 1 2977, 3597, 4217 70 argininosuccinate372394 219 361471 834 1738, 2358, synthase 1 2978, 3598, 4218 71argininosuccinate 422569 220 394212 835 1405, 1739, synthase 1 2359,2979, 3599, 4219 72 argininosuccinate 443588 221 397785 836 1406, 1740,synthase 1 2360, 2980, 3600, 4220 73 artemin 372354 222 361429 837 1741,2361, 2981, 3601, 4221, 4801, 5131, 6964-7050 74 artemin 372359 223361434 838 1742, 2362, 2982, 3602, 4222, 4802, 5132 75 artemin 414809224 387435 839 1743, 2363, 2983, 3603, 4223, 4803, 5133 76 artemin471394 225 435804 840 1744, 2364, 2984, 3604, 4224, 4804, 5134 77artemin 474592 226 434856 841 1745, 2365, 2985, 3605, 4225, 4805, 513578 artemin 477048 227 434784 842 1746, 2366, 2986, 3606, 4226, 4806,5136 79 artemin 491846 228 436149 843 1747, 2367, 2987, 3607, 4227,4807, 5137 80 artemin 498139 229 436727 844 1748, 2368, 2988, 3608,4228, 4808, 5138 81 arylsulfatase B 264914 230 264914 845 1749, 2369,1617 2989, 3609, 4229, 4809, 5139, 7051-7137 82 arylsulfatase B 264914231 264914 846 1750, 2370, 1617 2990, 3610, 4230, 4810, 5140 83arylsulfatase B 396151 232 379455 847 1751, 2371, 1617 2991, 3611, 4231,4811, 5141 84 arylsulfatase B 521117 233 428611 848 1752, 2372, 16172992, 3612, 4232, 4812, 5142 85 asparaginase 299234 234 299234 849 1753,2373, homolog (S. cerevisiae) 2993, 3613, 4233, 4813, 5143 86asparaginase 455920 235 389003 850 1754, 2374, homolog (S. cerevisiae)2994, 3614, 4234, 4814, 5144 87 asparaginase 550583 236 446856 851 1755,2375, homolog (S. cerevisiae) 2995, 3615, 4235, 4815, 5145 88asparaginase 551177 237 450040 852 1756, 2376, homolog (S. cerevisiae)2996, 3616, 4236, 4816, 5146 89 aspartoacylase 263080 238 263080 8531757, 2377, 2997, 3617, 4237 90 aspartoacylase 456349 239 409976 8541758, 2378, 2998, 3618, 4238, 7138-7224 91 ATPase, 283684 240 283684 8551407, 1759, aminophospholipid 2379, 2999, transporter, class 3619, 4239,I, type 8B, 7225-7398 member 1 92 ATPase, 536015 241 445359 856 1760,2380, aminophospholipid 3000, 3620, transporter, class 4240 I, type 8B,member 1 93 ATPase, Cu++ 242839 242 242839 857 1761, 2381, 1618transporting, beta 3001, 3621, polypeptide 4241, 4817, 5147, 7399-748594 ATPase, Cu++ 344297 243 342559 858 1762, 2382, 1618 transporting,beta 3002, 3622, polypeptide 4242, 4818, 5148 95 ATPase, Cu++ 400366 244383217 859 1763, 2383, 1618 transporting, beta 3003, 3623, polypeptide4243, 4819, 5149 96 ATPase, Cu++ 400370 245 383221 860 1764, 2384, 1618transporting, beta 3004, 3624, polypeptide 4244, 4820, 5150 97 ATPase,Cu++ 417240 246 390360 861 1765, 2385, transporting, beta 3005, 3625,polypeptide 4245, 4821, 5151 98 ATPase, Cu++ 418097 247 393343 862 1766,2386, 1618 transporting, beta 3006, 3626, polypeptide 4246, 4822, 515299 ATPase, Cu++ 448424 248 416738 863 1767, 2387, 1618 transporting,beta 3007, 3627, polypeptide 4247, 4823, 5153 100 ATPase, Cu++ 542656249 443128 864 1768, 2388, transporting, beta 3008, 3628, polypeptide4248, 4824, 5154 101 ATP-binding 263817 250 263817 865 1408, 1769,cassette, sub- 2389, 3009, family B 3629, 4249 (MDR/TAP), member 11 102ATP-binding 265723 251 265723 866 1409, 1770, cassette, sub- 2390, 3010,family B 3630, 4250, (MDR/TAP), 7486-7659 member 4 103 ATP-binding358400 252 351172 867 1410, 1771, cassette, sub- 2391, 3011, family B3631, 4251 (MDR/TAP), member 4 104 ATP-binding 359206 253 352135 8681411, 1772, cassette, sub- 2392, 3012, family B 3632, 4252 (MDR/TAP),member 4 105 ATP-binding 417608 254 394511 869 1412, 1773, cassette,sub- 2393, 3013, family B 3633, 4253 (MDR/TAP), member 4 106 ATP-binding453593 255 392983 870 1774, 2394, cassette, sub- 3014, 3634, family B4254 (MDR/TAP), member 4 107 ATP-binding 545634 256 437465 871 1775,2395, cassette, sub- 3015, 3635, family B 4255 (MDR/TAP), member 4 108bactericidal/permeability- 262865 257 262865 872 1776, 2396, increasing3016, 3636, protein 4256, 7660-7746 109 basic helix-loop- 242728 258242728 873 1413, 1777, helix family, 2397, 3017, member e41 3637, 4257,7747-7833 110 basic helix-loop- 540731 259 437369 874 1778, 2398, helixfamily, 3018, 3638, member e41 4258 111 bone 378827 260 368104 875 1414,1779, morphogenetic 2399, 3019, protein 2 3639, 4259, 7834-7920 112 bone395863 261 379204 876 1415, 1780, morphogenetic 2400, 3020, protein 73640, 4260, 4825, 5155, 7921-8007 113 bone 395864 262 379205 877 1416,1781, morphogenetic 2401, 3021, protein 7 3641, 4261, 4826, 5156 114Cap18 115 carbamoyl- 233072 263 233072 878 1417, 1782, phosphate 2402,3022, synthase 1, 3642, 4262 mitochondrial 116 carbamoyl- 417946 264388496 879 1418, 1783, phosphate 2403, 3023, synthase 1, 3643, 4263mitochondrial 117 carbamoyl- 430249 265 402608 880 1419, 1784, phosphate2404, 3024, synthase 1, 3644, 4264, mitochondrial 8008-8146 118carbamoyl- 451903 266 406136 881 1420, 1785, phosphate 2405, 3025,synthase 1, 3645, 4265 mitochondrial 119 carbamoyl- 518043 267 430697882 1786, 2406, phosphate 3026, 3646, synthase 1, 4266, mitochondrial120 carbamoyl- 536125 268 445539 883 1787, 2407, phosphate 3027, 3647,synthase 1, 4267 mitochondrial 121 carbamoyl- 539150 269 444139 8841788, 2408, phosphate 3028, 3648, synthase 1, 4268 mitochondrial 122carbamoyl- 544169 270 442790 885 1789, 2409, phosphate 3029, 3649,synthase 1, 4269 mitochondrial 123 cardiotrophin 1 279804 271 279804 8861790, 2410, 3030, 3650, 4270, 8147-8233 124 cardiotrophin 1 395019 272378465 887 1791, 2411, 3031, 3651, 4271 125 cathelicidin 296435 273296435 888 1792, 2412, 1619 antimicrobial 3032, 3652, peptide 4272,4827, 5157, 8234-8407 126 cathelicidin 576243 274 458149 889 1793, 2413,antimicrobial 3033, 3653, peptide 4273, 4828, 5158 127 Cbp/p300- 372638275 361721 890 1421, 1794, interacting 2414, 3034, transactivator, 3654,4274, with Glu/Asp-rich 8408-8494 carboxy-terminal domain, 4 128ceruloplasmin 264613 276 264613 891 1422, 1795, 1620 (ferroxidase) 2415,3035, 3655, 4275, 4829, 5159, 8495-8581 129 Cheetah Program 2;hyaluronidase (human); insulin lispro 130 ciliary 361987 277 355370 8921796, 2416, neurotrophic 3036, 3656, factor 4276 131 coagulation factor311907 278 308541 893 1423, 1797, II (thrombin) 2417, 3037, 3657, 4277,4830, 5160, 8582-8668 132 coagulation factor 446804 279 406403 894 1798,2418, II (thrombin) 3038, 3658, 4278, 4831, 5161 133 coagulation factor530231 280 433907 895 1424, 1799, II (thrombin) 2419, 3039, 3659, 4279,4832, 5162 134 coagulation factor 334047 281 334145 896 1425, 1800, III2420, 3040, (thromboplastin, 3660, 4280, tissue factor) 8669-8755 135coagulation factor 370207 282 359226 897 1426, 1801, III 2421, 3041,(thromboplastin, 3661, 4281, tissue factor) 8756-8842 136 coagulationfactor 218099 283 218099 898 1427, 1802, 1621 IX 2422, 3042, 3662, 4282,8843-9016 137 coagulation factor 394090 284 377650 899 1428, 1803, 1621IX 2423, 3043, 3663, 4283, 4833, 5163 138 coagulation factor 367796 285356770 900 1429, 1804, V (proaccelerin, 2424, 3044, labile factor) 3664,4284 139 coagulation factor 367797 286 356771 901 1430, 1805, V(proaccelerin, 2425, 3045, labile factor) 3665, 4285, 9017-9155 140coagulation factor 546081 287 439664 902 1431, 1806, V (proaccelerin,2426, 3046, labile factor) 3666, 4286 141 coagulation factor 346342 288329546 903 1432, 1807, 1622 VII (serum 2427, 3047, prothrombin 3667,4287, conversion 4834, 5164 accelerator) 142 coagulation factor 375581289 364731 904 1433, 1808, 1622 VII (serum 2428, 3048, prothrombin 3668,4288, conversion 4835, 5165, accelerator) 9156-9242 143 coagulationfactor 541084 290 442051 905 1434, 1809, 1622 VII (serum 2429, 3049,prothrombin 3669, 4289, conversion 4836, 5166 accelerator) 144coagulation factor 330287 291 327895 906 1810, 2430, VIII procoagulant3050, 3670, component 4290, 4837, 5167 145 coagulation factor 360256 292353393 907 1435, 1811, 1623 VIII, procoagulant 2431, 3051, component3671, 4291, 4838, 5168 146 coagulation factor X 375551 293 364701 9081436, 1812, 2432, 3052, 3672, 4292, 4839, 5169 147 coagulation factor X375559 294 364709 909 1437, 1813, 2433, 3053, 3673, 4293, 4840, 5170,9243-9381 148 coagulation factor X 409306 295 387092 910 1438, 1814,2434, 3054, 3674, 4294, 4841, 5171 149 coagulation factor 264692 296264692 911 1439, 1815, 1624 XI 2435, 3055, 3675, 4295, 4842, 5172 150coagulation factor 403665 297 384957 912 1440, 1816, 1624 XI 2436, 3056,3676, 4296, 4843, 5173, 9382-9468 151 coagulation factor 452239 298397401 913 1441, 1817, XI 2437, 3057, 3677, 4297, 4844, 5174, 152coagulation factor 264870 299 264870 914 1442, 1818, XIII, A1 2438,3058, polypeptide 3678, 4298, 9469-9555 153 coagulation factor 414279300 413334 915 1443, 1819, XIII, A1 2439, 3059, polypeptide 3679, 4299154 coagulation factor 441301 301 416127 916 1820, 2440, XIII, A1 3060,3680, polypeptide 4300 155 collagen, type VII, 328333 302 332371 9171821, 2441, 1625 alpha 1 3061, 3681, 4301, 4845, 5175, 9556-9642 156collagen, type VII, 454817 303 412569 918 1822, 2442, alpha 1 3062,3682, 4302, 4846, 5176, 157 colony stimulating 296871 304 296871 9191823, 2443, 1626 factor 2 3063, 3683, (granulocyte- 4303, 4847,macrophage) 5177, 9643-9781 158 colony stimulating 225474 305 225474 9201824, 2444, 1627 factor 3 3064, 3684, (granulocyte) 4304, 9782-9868 159colony stimulating 331769 306 327766 921 1825, 2445, factor 3 3065,3685, (granulocyte) 4305, 4848, 5178, 9869-9955 160 colony stimulating394148 307 377704 922 1826, 2446, 1627 factor 3 3066, 3686,(granulocyte) 4306, 4849, 5179, 161 colony stimulating 394149 308 377705923 1827, 2447, 1627 factor 3 3067, 3687, (granulocyte) 4307, 4850,5180, 9956-10042 162 complement factor H 367429 309 356399 924 1444,1828, 1627 2448, 3068, 3688, 4308, 4851, 5181 163 complement factor H391986 310 375846 925 1829, 2449, 3069, 3689, 4309, 4852, 5182 164complement factor H 439155 311 402656 926 1445, 1830, 2450, 3070, 3690,4310, 4853, 5183 165 copper metabolism 311832 312 308236 927 1446, 1831,(Murr1) domain 2451, 3071, containing 1 3691, 4311, 10043-10129 166copper metabolism 427417 313 413207 928 1447, 1832, (Murr1) domain 2452,3072, containing 1 3692, 4312 167 copper metabolism 444166 314 410050929 1448, 1833, (Murr1) domain 2453, 3073, containing 1 3693, 4313 168copper metabolism 458337 315 401236 930 1834, 2454, (Murr1) domain 3074,3694, containing 1 4314 169 copper metabolism 538736 316 438961 9311449, 1835, (Murr1) domain 2455, 3075, containing 1 3695, 4315 170corticotropin 276571 317 276571 932 1836, 2456, releasing hormone 3076,3696, 4316, 4854, 5184 171 CTLA4-Ig (Orencia) 172 cystic fibrosis 3084318 3084 933 1450, 1837, 1628 transmembrane 2457, 3077, conductance3697, 4317, regulator (ATP- 4855, 5185, binding cassette 10130-10216sub-family C, member 7) 173 cystic fibrosis 426809 319 389119 934 1451,1838, transmembrane 2458, 3078, conductance 3698, 4318, regulator (ATP-4856, 5186 binding cassette sub-family C, member 7) 174 cystic fibrosis454343 320 403677 935 1452, 1839, 1628 transmembrane 2459, 3079,conductance 3699, 4319, regulator (ATP- 4857, 5187 binding cassettesub-family C, member 7) 175 cystic fibrosis 468795 321 419254 936 1453,1840, transmembrane 2460, 3080, conductance 3700, 4320, regulator (ATP-4858, 5188 binding cassette sub-family C, member 7) 176 cystinosin381870 322 371294 937 1841, 2461, lysosomal cystine 3081, 3701,transporter 4321 177 cystinosin, 46640 323 46640 938 1454, 1842,lysosomal cystine 2462, 3082, transporter 3702, 4322, 4859, 5189,10217-10355 178 cystinosin, 414524 324 395471 939 1455, 1843, lysosomalcystine 2463, 3083, transporter 3703, 4323 179 cystinosin, 441220 325411465 940 1456, 1844, lysosomal cystine 2464, 3084, transporter 3704,4324 180 cystinosin, 452111 326 408652 941 1457, 1845, lysosomal cystine2465, 3085, transporter 3705, 4325 181 defensin, beta 314357 327 320951942 1846, 2466, 1629 103A 3086, 3706, 4326, 4860, 5190 182 defensin,beta 318124 328 324633 943 1847, 2467, 1630 103B 3087, 3707, 4327, 4861,5191, 10356-10442 183 defensin, beta 4A 302247 329 303532 944 1848,2468, 1631 3088, 3708, 4328, 4862, 5192, 10443-10529 184 defensin, beta4B 318157 330 424598 945 1849, 2469, 1632 3089, 3709, 4329, 4863, 5193185 DegludecPlus; insulin aspart; insulin degludec 186 deoxyribonucleaseI 246949 331 246949 946 1850, 2470, 1633 3090, 3710, 4330, 4864, 5194,10530-10616 187 deoxyribonuclease I 407479 332 385905 947 1851, 2471,1633 3091, 3711, 4331, 4865, 5195 188 deoxyribonuclease I 414110 333416699 948 1852, 2472, 3092, 3712, 4332, 4866, 5196 189 dysferlin, limb258104 334 258104 949 1458, 1853, girdle muscular 2473, 3093, dystrophy2B 3713, 4333, (autosomal 4867, 5197 recessive) 190 dysferlin, limb394120 335 377678 950 1459, 1854, 1634 girdle muscular 2474, 3094,dystrophy 2B 3714, 4334, (autosomal 4868, 5198, recessive) 10617-10703191 dysferlin, limb 409366 336 386512 951 1460, 1855, girdle muscular2475, 3095, dystrophy 2B 3715, 4335, (autosomal 4869, 5199 recessive)192 dysferlin, limb 409582 337 386547 952 1461, 1856, girdle muscular2476, 3096, dystrophy 2B 3716, 4336, (autosomal 4870, 5200 recessive)193 dysferlin, limb 409651 338 386683 953 1462, 1857, girdle muscular2477, 3097, dystrophy 2B 3717, 4337, (autosomal 4871, 5201 recessive)194 dysferlin, limb 409744 339 386285 954 1463, 1858, girdle muscular2478, 3098, dystrophy 2B 3718, 4338, (autosomal 4872, 5202 recessive)195 dysferlin, limb 409762 340 387137 955 1464, 1859, girdle muscular2479, 3099, dystrophy 2B 3719, 4339, (autosomal 4873, 5203 recessive)196 dysferlin, limb 410020 341 386881 956 1465, 1860, girdle muscular2480, 3100, dystrophy 2B 3720, 4340, (autosomal 4874, 5204 recessive)197 dysferlin, limb 410041 342 386617 957 1466, 1861, girdle muscular2481, 3101, dystrophy 2B 3721, 4341, (autosomal 4875, 5205 recessive)198 dysferlin, limb 413539 343 407046 958 1467, 1862, girdle muscular2482, 3102, dystrophy 2B 3722, 4342, (autosomal 4876, 5206 recessive)199 dysferlin, limb 429174 344 398305 959 1468, 1863, girdle muscular2483, 3103, dystrophy 2B 3723, 4343, (autosomal 4877, 5207 recessive)200 ectodysplasin A 338901 345 340611 960 1864, 2484, 3104, 3724, 4344,4878, 5208 201 ectodysplasin A 374552 346 363680 961 1865, 2485, 3105,3725, 4345, 4879, 5209 202 ectodysplasin A 374553 347 363681 962 1866,2486, 1635 3106, 3726, 4346, 4880, 5210, 10704-10790 203 ectodysplasin A525810 348 434195 963 1867, 2487, 3107, 3727, 4347, 4881, 5211 204ectodysplasin A 524573 349 432585 964 1868, 2488, 3108, 3728, 4348,4882, 5212 205 ectodysplasin A 527388 350 434861 965 1869, 2489, 3109,3729, 4349, 4883, 5213 206 enamelin 396073 351 379383 966 1870, 2490,1636 3110, 3730, 4350, 4884, 5214 207 erythropoietin 252723 352 252723967 1871, 2491, 1637 3111, 3731, 4351, 4885, 5215, 10791-11051 208Exenatide 968 1872, 2492, 3112, 3732, 4352, 4886, 5216 209 family with252530 353 252530 969 1873, 2493, sequence 3113, 3733, similarity 98,4353 member C 210 family with 343358 354 340348 970 1874, 2494, sequence3114, 3734, similarity 98, 4354 member C 211 fibrinogen alpha 302053 355306361 971 1469, 1875, chain 2495, 3115, 3735, 4355, 11052-11190 212fibrinogen alpha 403106 356 385981 972 1470, 1876, chain 2496, 3116,3736, 4356 213 fibrinogen alpha 457487 357 407891 973 1877, 2497, chain3117, 3737, 4357 214 fibrinogen beta 302068 358 306099 974 1471, 1878,chain 2498, 3118, 3738, 4358, 11191-11329 215 fibrinogen beta 537843 359437727 975 1879, 2499, chain 3119, 3739, 4359 216 fibrinogen gamma336098 360 336829 976 1472, 1880, chain 2500, 3120, 3740, 4360,11330-11468 217 fibrinogen gamma 404648 361 384860 977 1473, 1881, chain2501, 3121, 3741, 4361 218 fibroblast growth 222157 362 222157 978 1474,1882, factor 21 2502, 3122, 3742, 4362 219 fibroblast growth 593756 363471477 979 1883, 2503, factor 21 3123, 3743, 4363, 11469-11555 220fibronectin type III 373470 364 362569 980 1884, 2504, domain containing5 3124, 3744, 4364, 221 fibronectin type III 373471 365 362570 981 1885,2505, domain containing 5 3125, 3745, 4365, 11556-11642 222 folliclestimulating 254122 366 254122 982 1886, 2506, hormone, beta 3126, 3746,polypeptide 4366, 4887, 5217, 11643-11729 223 follicle stimulating417547 367 416606 983 1887, 2507, hormone, beta 3127, 3747, polypeptide4367, 4888, 5218 224 follicle stimulating 533718 368 433424 984 1888,2508, hormone, beta 3128, 3748, polypeptide 4368, 4889, 5219 225fumarylacetoacetate 261755 369 261755 985 1475, 1889, hydrolase 2509,3129, (fumarylacetoacetase) 3749, 4369 226 fumarylacetoacetate 407106370 385080 986 1890, 2510, hydrolase 3130, 3750, (fumarylacetoacetase)4370, 11730-11868 227 fumarylacetoacetate 561421 371 453347 987 1891,2511, hydrolase 3131, 3751, (fumarylacetoacetase) 4371 228 galactokinase1 225614 372 225614 988 1892, 2512, 3132, 3752, 4372 229 galactokinase 1375188 373 364334 989 1893, 2513, 3133, 3753, 4373 230 galactokinase 1437911 374 406305 990 1894, 2514, 3134, 3754, 4374 231 galactokinase 1588479 375 465930 991 1895, 2515, 3135, 3755, 4375, 11869-11955 232galactosamine (N- 268695 376 268695 992 1476, 1896, 1638acetyl)-6-sulfate 2516, 3136, sulfatase 3756, 4376, 4890, 5220,11956-12042 233 galactosamine (N- 439266 377 402127 993 1897, 2517,acetyl)-6-sulfate 3137, 3757, sulfatase 4377, 4891, 5221 234galactosamine (N- 542788 378 438197 994 1477, 1898, acetyl)-6-sulfate2518, 3138, sulfatase 3758, 4378, 4892, 5222 235 galactose-1- 378842 379368119 995 1899, 2519, phosphate 3139, 3759, uridylyltransferase 4379,12043-12181 236 galactose-1- 450095 380 401956 996 1900, 2520, phosphate3140, 3760, uridylyltransferase 4380 237 galactose-1- 554550 381 451435997 1901, 2521, phosphate 3141, 3761, uridylyltransferase 4381 238galactosidase, 218516 382 218516 998 1902, 2522, 1639 alpha 3142, 3762,4382, 12182-12268 239 Galectin 3 383 999 1640 inhibitor (A9) 240Galectin 3 384 1000 1641 inhibitor (C12) 241 glial cell derived 326524385 317145 1001 1903, 2523, neurotrophic 3143, 3763, factor 4383, 4893,5223 242 glial cell derived 344622 386 339703 1002 1904, 2524,neurotrophic 3144, 3764, factor 4384, 4894, 5224 243 glial cell derived427982 387 409007 1003 1905, 2525, 1642 neurotrophic 3145, 3765, factor4385, 4895, 5225, 12269-12355 244 glial cell derived 502572 388 4235571004 1906, 2526, neurotrophic 3146, 3766, factor 4386, 4896, 5226 245glial cell derived 510177 389 424592 1005 1907, 2527, neurotrophic 3147,3767, factor 4387, 4897, 5227 246 glial cell derived 515058 390 4259281006 1908, 2528, neurotrophic 3148, 3768, factor 4388, 4898, 5228 247glial cell derived 381826 391 371248 1007 1909, 2529, neurotrophic 3149,3769, factor 4389, 4899, 5229 248 GLP-1 Fc; dulaglutide 249 glucagon375497 392 364647 1008 1910, 2530, 1643 3150, 3770, 4390, 4900, 5230 250glucagon 418842 393 387662 1009 1911, 2531, 1643 3151, 3771, 4391, 4901,5231, 12356-12703 251 glucan (1,4-alpha-), 264326 394 264326 1010 1912,2532, branching 3152, 3772, enzyme 1 4392 252 glucan (1,4-alpha-),429644 395 410833 1011 1478, 1913, branching 2533, 3153, enzyme 1 3773,4393, 12704-12842 253 glucan (1,4-alpha-), 536832 396 445365 1012 1914,2534, branching 3154, 3774, enzyme 1 4394 254 glucose-6- 253801 397253801 1013 1915, 2535, phosphatase, 3155, 3775, catalytic subunit 4395,12843-12981 255 glucosidase, alpha; 302262 398 305692 1014 1916, 2536,1644 acid 3156, 3776, 4396, 4902, 5232, 12982-13155 256 glucosidase,alpha; 390015 399 374665 1015 1917, 2537, 1644 acid 3157, 3777, 4397,4903, 5233 257 glucosidase, beta, 327247 400 314508 1016 1918, 2538,1645 acid 3158, 3778, 4398, 4904, 5234 258 glucosidase, beta, 368373 401357357 1017 1919, 2539, 1645 acid 3159, 3779, 4399, 4905, 5235 259glucosidase, beta, 402928 402 385813 1018 1920, 2540, 1645 acid 3160,3780, 4400, 4906, 5236 260 glucosidase, beta, 427500 403 402577 10191921, 2541, 1645 acid 3161, 3781, 4401, 4907, 5237 261 glucosidase,beta, 428024 404 397986 1020 1922, 2542, 1645 acid 3162, 3782, 4402,4908, 5238 262 glucosidase, beta, 536555 405 446457 1021 1923, 2543,1645 acid 3163, 3783, 4403, 4909, 5239 263 glucosidase, beta, 536770 406445560 1022 1924, 2544, 1645 acid 3164, 3784, 4404, 4910, 5240 264glycogen synthase 261195 407 261195 1023 1925, 2545, 2 (liver) 3165,3785, 4405, 13156-13242 265 glycoprotein 369582 408 358595 1024 1926,2546, 1646 hormones, alpha 3166, 3786, polypeptide 4406, 4911, 5241,13243-13329 266 GP2013; rituximab 267 Granulysin 9 kDa 1479(non-secreted) 268 Granulysin 9 kDa 1480 (secreted) 269 growth hormone 1323322 409 312673 1025 1927, 2547, 1647 3167, 3787, 4407, 4912, 5242,13330-13416 270 growth hormone 1 342364 410 339278 1026 1928, 2548, 16473168, 3788, 4408, 4913, 5243 271 growth hormone 1 351388 411 343791 10271929, 2549, 1647 3169, 3789, 4409, 4914, 5244 272 growth hormone 1458650 412 408486 1028 1930, 2550, 1647 3170, 3790, 4410, 4915, 5245 273growth hormone 2 332800 413 333157 1029 1931, 2551, 3171, 3791, 4411,4916, 5246 274 growth hormone 2 423893 414 409294 1030 1932, 2552, 3172,3792, 4412, 4917, 5247 275 growth hormone 2 449787 415 410618 1031 1933,2553, 3173, 3793, 4413, 4918, 5248 276 growth hormone 2 456543 416394122 1032 1934, 2554, 3174, 3794, 4414, 4919, 5249 277 GTP 395514 417378890 1033 1935, 2555, 1648 cyclohydrolase 1 3175, 3795, 4415, 4920,5250, 13417-13503 278 GTP 395524 418 378895 1034 1936, 2556,cyclohydrolase 1 3176, 3796, 4416, 4921, 5251 279 GTP 491895 419 4190451035 1937, 2557, 1649 cyclohydrolase 1 3177, 3797, 4417, 4922, 5252 280GTP 536224 420 445246 1036 1938, 2558, cyclohydrolase 1 3178, 3798,4418, 4923, 5253 281 GTP 543643 421 444011 1037 1939, 2559,cyclohydrolase 1 3179, 3799, 4419, 4924, 5254 282 hemochromatosis 309234422 311698 1038 1940, 2560, 3180, 3800, 4420 283 hemochromatosis 317896423 313776 1039 1941, 2561, 3181, 3801, 4421 284 hemochromatosis 336625424 337819 1040 1942, 2562, 3182, 3802, 4422 285 hemochromatosis 349999425 259699 1041 1943, 2563, 3183, 3803, 4423 286 hemochromatosis 352392426 315936 1042 1944, 2564, 3184, 3804, 4424 287 hemochromatosis 353147427 312342 1043 1945, 2565, 3185, 3805, 4425 288 hemochromatosis 357618428 417404 1044 1946, 2566, 3186, 3806, 4426 289 hemochromatosis 397022429 380217 1045 1947, 2567, 3187, 3807, 4427 290 hemochromatosis 461397430 420802 1046 1948, 2568, 3188, 3808, 4428 291 hemochromatosis 470149431 419725 1047 1949, 2569, 3189, 3809, 4429 292 hemochromatosis 488199432 420559 1048 1950, 2570, 3190, 3810, 4430 293 hemochromatosis 535098433 445872 1049 1951, 2571, 3191, 3811, 4431 294 hemochromatosis 539147434 445098 1050 1952, 2572, 3192, 3812, 4432 295 hemochromatosis 336751435 337014 1051 1481, 1953, type 2 (juvenile) 2573, 3193, 3813, 4433,4925, 5255, 13504-13590 296 hemochromatosis 357836 436 350495 1052 1482,1954, type 2 (juvenile) 2574, 3194, 3814, 4434, 4926, 5256 297hemochromatosis 421822 437 411863 1053 1483, 1955, type 2 (juvenile)2575, 3195, 3815, 4435, 4927, 5257 298 hemochromatosis 475797 438 4257161054 1484, 1956, type 2 (juvenile) 2576, 3196, 3816, 4436, 4928, 5258299 hemochromatosis 497365 439 421820 1055 1957, 2577, type 2 (juvenile)3197, 3817, 4437, 4929, 5259 300 hemochromatosis 577520 440 463276 10561958, 2578, type 2 (juvenile) 3198, 3818, 4438 301 hemochromatosis580693 441 464413 1057 1959, 2579, type 2 (juvenile) 3199, 3819, 4439302 hepatocyte growth 222390 442 222390 1058 1960, 2580, factor(hepapoietin 3200, 3820, A; scatter factor) 4440, 13591-13677 303hepatocyte growth 354224 443 346164 1059 1961, 2581, factor (hepapoietin3201, 3821, A; scatter factor) 4441 304 hepatocyte growth 394769 444378250 1060 1962, 2582, factor (hepapoietin 3202, 3822, A; scatterfactor) 4442 305 hepatocyte growth 412881 445 396307 1061 1963, 2583,factor (hepapoietin 3203, 3823, A; scatter factor) 4443 306 hepatocytegrowth 421558 446 388592 1062 1964, 2584, factor (hepapoietin 3204,3824, A; scatter factor) 4444 307 hepatocyte growth 423064 447 4138291063 1965, 2585, factor (hepapoietin 3205, 3825, A; scatter factor) 4445308 hepatocyte growth 444829 448 389854 1064 1966, 2586, factor(hepapoietin 3206, 3826, A; scatter factor) 4446 309 hepatocyte growth453411 449 408270 1065 1967, 2587, factor (hepapoietin 3207, 3827, A;scatter factor) 4447 310 hepatocyte growth 457544 450 391238 1066 1968,2588, factor (hepapoietin 3208, 3828, A; scatter factor) 4448 311hepcidin 222304 451 222304 1067 14851969, antimicrobial 2589, 3209,peptide 3829, 4449 312 hepcidin 598398 452 471894 1068 1970, 2590,antimicrobial 3210, 3830, peptide 4450, 13678-13764 313 Herceptin Heavy1069 1971, 2591, Chain 3211, 3831, 4451, 4930, 5260 314 Herceptin Light1070 1972, 2592, Chain 3212, 3832, 4452, 4931, 5261 315 hypoxanthine298556 453 298556 1071 1486, 1973, 1650 phosphoribosyltransferase 12593, 3213, 3833, 4453, 4932, 5262, 13765-13851 316 hypoxanthine 370796454 359832 1072 1974, 2594, phosphoribosyltransferase 1 3214, 3834,4454, 4933, 5263 317 IDegLira; insulin degludec; liraglutide 318iduronate 2- 340855 455 339801 1073 1975, 2595, 1651 sulfatase 3215,3835, 4455, 4934, 5264 319 iduronate 2- 370441 456 359470 1074 1976,2596, 1651 sulfatase 3216, 3836, 4456, 4935, 5265 320 iduronate 2-370443 457 359472 1075 1977, 2597, 1651 sulfatase 3217, 3837, 4457,4936, 5266 321 iduronate 2- 428056 458 390241 1076 1978, 2598, 1651sulfatase 3218, 3838, 4458, 4937, 5267 322 iduronate 2- 521702 459429745 1077 1979, 2599, 1651 sulfatase 3219, 3839, 4459, 4938, 5268 323iduronate 2- 537071 460 440324 1078 1980, 2600, 1651 sulfatase 3220,3840, 4460, 4939, 5269 324 iduronate 2- 541269 461 441261 1079 1981,2601, 1651 sulfatase 3221, 3841, 4461, 4940, 5270 325 iduronidase,alpha- 247933 462 247933 1080 1982, 2602, 1652 L- 3222, 3842, 4462,4941, 5271, 13852-13938 326 iduronidase, alpha- 453894 463 396458 10811983, 2603, 1652 L- 3223, 3843, 4463, 4942, 5272 327 IgM Heavy 328 IgMLight 329 INS-IGF2 397270 464 380440 1082 1984, 2604, readthrough 3224,3844, 4464, 4943, 5273 330 insulin 250971 465 250971 1083 1985, 2605,1653 3225, 3845, 4465, 4944, 5274, 13939-14286 331 insulin 381330 466370731 1084 1986, 2606, 1653 3226, 3846, 4466, 4945, 5275 332 insulin397262 467 380432 1085 1987, 2607, 1653 3227, 3847, 4467, 4946, 5276,14287-14373 333 insulin 512523 468 424008 1086 1988, 2608, 3228, 3848,4468, 4947, 5277 334 Insulin Aspart 1087 1989, 2609, 3229, 3849, 4469,4948, 5278 335 Insulin Glargine 1088 1990, 2610, 3230, 3850, 4470, 4949,5279 336 Insulin Glulisine 1089 1991, 2611, 3231, 3851, 4471, 4950, 5280337 Insulin Lispro 1090 1992, 2612, 3232, 3852, 4472, 4951, 5281 338interferon, alpha 357374 469 369566 1091 1993, 2613, 10 3233, 3853,4473, 4952, 5282 339 interferon, alpha 449498 470 394494 1092 1994,2614, 13 3234, 3854, 4474, 4953, 5283 340 interferon, alpha 380222 471369571 1093 1995, 2615, 14 3235, 3855, 4475, 4954, 5284 341 interferon,alpha 380216 472 369564 1094 1996, 2616, 16 3236, 3856, 4476, 4955, 5285342 interferon, alpha 413767 473 411940 1095 1997, 2617, 17 3237, 3857,4477, 4956, 5286 343 interferon, alpha 2 380206 474 369554 1096 1998,2618, 1654 3238, 3858, 4478, 4957, 5287, 14374-14460 344 interferon,alpha 380225 475 369574 1097 1999, 2619, 21 3239, 3859, 4479, 4958, 5288345 interferon, alpha 4 421715 476 412897 1098 2000, 2620, 3240, 3860,4480, 4959, 5289 346 interferon, alpha 5 259555 477 259555 1099 2001,2621, 3241, 3861, 4481, 4960, 5290 347 interferon, alpha 6 380210 478369558 1100 2002, 2622, 3242, 3862, 4482, 4961, 5291 348 interferon,alpha 7 239347 479 239347 1101 2003, 2623, 3243, 3863, 4483, 4962, 5292349 interferon, alpha 8 380205 480 369553 1102 2004, 2624, 3244, 3864,4484, 4963, 5293 350 interferon, beta 1, 380232 481 369581 1103 2005,2625, 1655 fibroblast 3245, 3865, 4485, 4964, 5294, 14461-14547 351interleukin 10 423557 482 412237 1104 1487, 2006, 2626, 3246, 3866,4486, 14548-14634 352 interleukin 21 264497 483 264497 1105 2007, 2627,3247, 3867, 4487, 14635-14721 353 interleukin 7 263851 484 263851 11061488, 2008, 1656 2628, 3248, 3868, 4488, 4965, 5295, 14722-14860 354interleukin 7 379113 485 368408 1107 1489, 2009, 1656 2629, 3249, 3869,4489, 4966, 5296 355 interleukin 7 379114 486 368409 1108 2010, 2630,3250, 3870, 4490, 4967, 5297 356 interleukin 7 518982 487 430272 11092011, 2631, 3251, 3871, 4491, 4968, 5298 357 interleukin 7 520215 488428364 1110 1490, 2012, 2632, 3252, 3872, 4492, 4969, 5299 358interleukin 7 520269 489 427750 1111 1491, 2013, 2633, 3253, 3873, 4493,4970, 5300 359 interleukin 7 520317 490 427800 1112 2014, 2634, 3254,3874, 4494, 4971, 5301 360 interleukin 7 541183 491 438922 1113 1492,2015, 2635, 3255, 3875, 4495, 4972, 5302 361 KIT ligand 228280 492228280 1114 2016, 2636, 3256, 3876, 4496, 14861-14947 362 KIT ligand347404 493 54216 1115 2017, 2637, 3257, 3877, 4497 363 KIT ligand 537835494 438889 1116 2018, 2638, 3258, 3878, 4498 364 klotho 380099 495369442 1117 1493, 2019, 2639, 3259, 3879, 4499, 14948-15347 365 klotho426690 496 399513 1118 14942020, 2640, 3260, 3880, 4500 366 Kruppel-like374672 497 363804 1119 2021, 2641, factor 4 (gut) 3261, 3881, 4501 367Kruppel-like 411706 498 399921 1120 2022, 2642, factor 4 (gut) 3262,3882, 4502 368 Kruppel-like 420475 499 404922 1121 2023, 2643, factor 4(gut) 3263, 3883, 4503 369 Kruppel-like 439281 500 396294 1122 2024,2644, factor 4 (gut) 3264, 3884, 4504 370 lecithin- 264005 501 2640051123 1495, 2025, cholesterol 2645, 3265, acyltransferase 3885, 4505,15348-15434 371 lectin, galactoside- 254301 502 254301 1124 2026, 2646,binding, soluble, 3 3266, 3886, 4506, 15435-15521 372 lin-28 homolog A254231 503 254231 1125 2027, 2647, (C. elegans) 3267, 3887, 4507 373lin-28 homolog A 326279 504 363314 1126 2028, 2648, (C. elegans) 3268,3888, 4508, 15522-15608 374 lipase A, 282673 505 282673 1127 1496, 2029,lysosomal acid, 2649, 3269, cholesterol 3889, 4509, esterase 4973, 5303375 lipase A, 336233 506 337354 1128 1497, 2030, 1657 lysosomal acid,2650, 3270, cholesterol 3890, 4510, esterase 4974, 5304, 15609-15782 376lipase A, 354621 507 360895 1129 2031, 2651, lysosomal acid, 3271, 3891,cholesterol 4511, 4975, esterase 5305 377 lipase A, 371829 508 3608941130 2032, 2652, 1657 lysosomal acid, 3272, 3892, cholesterol 4512,4976, esterase 5306 378 lipase A, 371837 509 360903 1131 1498, 2033,lysosomal acid, 2653, 3273, cholesterol 3893, 4513, esterase 4977, 5307379 lipase A, 425287 510 392037 1132 1499, 2034, lysosomal acid, 2654,3274, cholesterol 3894, 4514, esterase 4978, 5308 380 lipase A, 428800511 388415 1133 1500, 2035, 1657 lysosomal acid, 2655, 3275, cholesterol3895, 4515, esterase 4979, 5309 381 lipase A, 456827 512 413019 11342036, 2656, 1657 lysosomal acid, 3276, 3896, cholesterol 4516, 4980,esterase 5310 382 lipase A, 540050 513 439727 1135 2037, 2657, lysosomalacid, 3277, 3897, cholesterol 4517, 4981, esterase 5311 383 lipase A,541980 514 438127 1136 2038, 2658, 1657 lysosomal acid, 3278, 3898,cholesterol 4518, 4982, esterase 5312 384 lipase A, 542307 515 4375641137 1501, 2039, 1657 lysosomal acid, 2659, 3279, cholesterol 3899,4519, esterase 4983, 5313 385 lipoprotein lipase 311322 516 309757 11381502, 2040, 2660, 3280, 3900, 4520, 4984, 5314, 15783-15869 386lipoprotein lipase 520959 517 428496 1139 1503, 2041, 2661, 3281, 3901,4521, 4985, 5315 387 lipoprotein lipase 522701 518 428557 1140 1504,2042, 2662, 3282, 3902, 4522, 4986, 5316 388 lipoprotein lipase 524029519 428237 1141 1505, 2043, 2663, 3283, 3903, 4523, 4987, 5317 389lipoprotein lipase 535763 520 438606 1142 2044, 2664, 3284, 3904, 4524,4988, 5318 390 lipoprotein lipase 538071 521 439407 1143 2045, 2665,3285, 3905, 4525, 4989, 5319 391 liraglutide native 1506 7-37 392liraglutide native 1507 7-37-A8G 393 liraglutide native 15087-37-A8G-10X 394 liraglutide; GLP1; 1509 Victoza 395 LL-37; 1144 1620Cathelicidin anti- microbial peptide 396 low density 455727 522 3978291145 1510, 2046, lipoprotein 2666, 3286, receptor 3906, 4526 397 lowdensity 535915 523 440520 1146 1511, 2047, lipoprotein 2667, 3287,receptor 3907, 4527 398 low density 545707 524 437639 1147 1512, 2048,lipoprotein 2668, 3288, receptor 3908, 4528 399 low density 558013 525453346 1148 1513, 2049, lipoprotein 2669, 3289, receptor 3909, 4529 400low density 558518 526 454071 1149 1514, 2050, lipoprotein 2670, 3290,receptor 3910, 4530, 15870-16478 401 low density 561343 527 454147 11502051, 2671, lipoprotein 3291, 3911, receptor 4531 402 low density 252444528 252444 1151 2052, 2672, lipoprotein 3292, 3912, receptor 4532 403luteinizing 221421 529 221421 1152 2053, 2673, hormone beta 3293, 3913,polypeptide 4533 404 luteinizing 391870 530 375743 1153 2054, 2674,hormone beta 3294, 3914, polypeptide 4534, 4990, 5320, 405 LY2963016;insulin glargine 406 Ly6/neurotoxin 1 317543 531 319846 1154 2055, 2675,3295, 3915, 4535 407 MabThera SC; rituximab 408 mannosidase alpha 221363532 221363 1155 2056, 2676, class 2B member 1 3296, 3916, 4536 409mannosidase, 433513 533 390583 1156 1515, 2057, alpha, class 2B, 2677,3297, member 1 3917, 4537 410 mannosidase, 456935 534 395473 1157 1516,2058, alpha, class 2B, 2678, 3298, member 1 3918, 4538, 16479-16565 411mannosidase, 536796 535 438266 1158 2059, 2679, alpha, class 2B, 3299,3919, member 1 4539 412 meteorin glial cell 568223 536 455068 1159 2060,2680, differentiation 3300, 3920, regulator 4540, 16566-16652 413meteorin, glial cell 219542 537 219542 1160 2061, 2681, differentiation3301, 3921, regulator 4541 414 methylmalonyl 274813 538 274813 11611517, 2062, 1658 CoA mutase 2682, 3302, 3922, 4542, 4991, 5321,16653-16739 415 methylmalonyl 540138 539 445535 1162 2063, 2683, CoAmutase 3303, 3923, 4543, 4992, 5322, 416 microsomal 265517 540 2655171163 1518, 2064, triglyceride 2684, 3304, transfer protein 3924, 4544,16740-16878 417 microsomal 457717 541 400821 1164 2065, 2685,triglyceride 3305, 3925, transfer protein 4545 418 microsomal 506883 542426755 1165 1519, 2066, triglyceride 2686, 3306, transfer protein 3926,4546 419 microsomal 538053 543 437358 1166 2067, 2687, triglyceride3307, 3927, transfer protein 4547 420 N- 299314 544 299314 1167 1520,2068, acetylglucosamine- 2688, 3308, 1-phosphate 3928, 4548,transferase, alpha 16879-17017 and beta subunits 421 N- 549940 545449150 1168 1521, 2069, acetylglucosamine- 2689, 3309, 1-phosphate 3929,4549 transferase, alpha and beta subunits 422 N-acetylglutamate 293404546 293404 1169 2070, 2690, synthase 3310, 3930, 4550, 17018-17156 423N-acetylglutamate 541745 547 441262 1170 2071, 2691, synthase 3311,3931, 4551 424 N-acylsphingosine 262097 548 262097 1171 1522, 2072,amidohydrolase 2692, 3312, (acid ceramidase) 1 3932, 455217157-17295 425N-acylsphingosine 314146 549 326970 1172 1523, 2073, amidohydrolase2693, 3313, (acid ceramidase) 1 3933, 4553 426 N-acylsphingosine 381733550 371152 1173 1524, 2074, amidohydrolase 2694, 3314, (acid ceramidase)1 3934, 4554 427 N-acylsphingosine 417108 551 394125 1174 1525, 2075,amidohydrolase 2695, 3315, (acid ceramidase) 1 3935, 4555 428N-acylsphingosine 520781 552 427751 1175 1526, 2076, amidohydrolase2696, 3316, (acid ceramidase) 1 3936, 4556 429 natriuretic peptide B376468 553 365651 1176 2077, 2697, 3317, 3937, 4557, 4993, 5323, 430nerve growth 369512 554 358525 1177 2078, 2698, factor (beta 3318, 3938,polypeptide) 4558, 4994, 5324, 17296-17382 431 neuregulin 1 287840 555287840 1178 2079, 2699, 3319, 3939, 4559 432 neuregulin 1 287842 556287842 1179 2080, 2700, 3320, 3940, 4560, 17383-17469 433 neuregulin 1287845 557 287845 1180 2081, 2701, 3321, 3941, 4561 434 neuregulin 1338921 558 343395 1181 2082, 2702, 3322, 3942, 4562 435 neuregulin 1341377 559 340497 1182 2083, 2703, 3323, 3943, 4563 436 neuregulin 1356819 560 349275 1183 2084, 2704, 3324, 3944, 4564 437 neuregulin 1405005 561 384620 1184 2085, 2705, 3325, 3945, 4565 438 neuregulin 1519301 562 429582 1185 2086, 2706, 3326, 3946, 4566 439 neuregulin 1520407 563 434640 1186 2087, 2707, 3327, 3947, 4567, 17470-17556 440neuregulin 1 520502 564 433289 1187 2088, 2708, 3328, 3948, 4568 441neuregulin 1 521670 565 428828 1188 2089, 2709, 3329, 3949, 4569 442neuregulin 1 539990 566 439276 1189 2090, 2710, 3330, 3950, 4570 443neuregulin 1 523079 567 430120 1190 2091, 2711, 3331, 3951, 4571 444OMOMYC 1191 1527, 17557-17643 445 OMOMYC90 1528, 17644-17784 (90AAtruncated domain) 446 Orencia S.C.; abatacept 447 ornithine 39007 56839007 1192 1529, 2092, 1659 carbamoyltransferase 2712, 3332, 3952, 4572,4995, 5325, 17785-17871 448 parathyroid 282091 569 282091 1193 2093,2713, hormone 3333, 3953, 4573, 4996, 5326 449 parathyroid 529816 570433208 1194 2094, 2714, hormone 3334, 3954, 4574, 4997, 5327 450PBO-326; rituximab 451 phenylalanine 546844 571 446658 1195 1530, 2095,hydroxylase 2715, 3335, 3955, 4575, 4998, 5328 452 phenylalanine 551337572 447620 1196 1531, 2096, hydroxylase 2716, 3336, 3956, 4576, 4999,5329 453 phenylalanine 553106 573 448059 1197 1532, 2097, 1660hydroxylase 2717, 3337, 3957, 4577, 5000, 5330, 17872-17958 454phosphorylase 566044 574 456729 1198 2098, 2718, kinase beta 3338, 3958,4578 455 phosphorylase 563588 575 455607 1199 2099, 2719, kinase gamma 23339, 3959, (testis) 4579, 17959-18045 456 phosphorylase 379942 576369274 1200 2100, 2720, kinase, alpha 2 3340, 3960, (liver) 4580,18046-18132 457 phosphorylase 299167 577 299167 1201 2101, 2721, kinase,beta 3341, 3961, 4581 458 phosphorylase 323584 578 313504 1202 2102,2722, kinase, beta 3342, 3962, 4582, 18133-18219 459 phosphorylase455779 579 414345 1203 2103, 2723, kinase, beta 3343, 3963, 4583 460phosphorylase 328273 580 329968 1204 2104, 2724, kinase, gamma 2 3344,3964, (testis) 4584 461 phosphorylase 424889 581 388571 1205 2105, 2725,kinase, gamma 2 3345, 3965, (testis) 4585 462 phosphorylase, 216392 582216392 1206 2106, 2726, glycogen, liver 3346, 3966, 4586 463phosphorylase, 544180 583 443787 1207 2107, 2727, glycogen, liver 3347,3967, 4587 464 plasminogen 308192 584 308938 1208 1533, 2108, 2728,3348, 3968, 4588, 18220-18271 465 plasminogen 316325 585 321466 12092109, 2729, 3349, 3969, 4589 466 plasminogen 366924 586 355891 12101534, 2110, 2730, 3350, 3970, 4590 467 plasminogen 220809 587 2208091211 1535, 2111, 1661 activator, tissue 2731, 3351, 3971, 4591,18272-18358 468 plasminogen 270189 588 270189 1212 1536, 2112, 1661activator, tissue 2732, 3352, 3972, 4592, 5001, 5331 469 plasminogen352041 589 270188 1213 1537, 2113, 1661 activator, tissue 2733, 3353,3973, 4593, 5002, 5332 470 plasminogen 429089 590 392045 1214 2114,2734, 1661 activator, tissue 3354, 3974, 4594, 5003, 5333 471plasminogen 429710 591 407861 1215 1538, 2115, activator, tissue 2735,3355, 3975, 4595, 5004, 5334 472 plasminogen 519510 592 428886 12161539, 2116, activator, tissue 2736, 3356, 3976, 4596, 5005, 5335 473plasminogen 520523 593 428797 1217 1540, 2117, 1661 activator, tissue2737, 3357, 3977, 4597, 5006, 5336 474 plasminogen 521694 594 4298011218 1541, 2118, 1661 activator, tissue 2738, 3358, 3978, 4598, 5007,5337 475 plasminogen 524009 595 429401 1219 1542, 2119, activator,tissue 2739, 3359, 3979, 4599, 5008, 5338 476 POU class 5 259915 596259915 1220 2120, 2740, homeobox 1 3360, 3980, 4600 477 POU class 5376243 597 365419 1221 2121, 2741, homeobox 1 3361, 3981, 4601 478 POUclass 5 383524 598 373016 1222 2122, 2742, homeobox 1 3362, 3982, 4602479 POU class 5 412166 599 387646 1223 2123, 2743, homeobox 1 3363,3983, 4603 480 POU class 5 419095 600 413622 1224 2124, 2744, homeobox 13364, 3984, 4604 481 POU class 5 429603 601 392877 1225 2125, 2745,homeobox 1 3365, 3985, 4605 482 POU class 5 433063 602 405041 1226 2126,2746, homeobox 1 3366, 3986, 4606 483 POU class 5 433348 603 412665 12272127, 2747, homeobox 1 3367, 3987, 4607 484 POU class 5 434616 604388842 1228 2128, 2748, homeobox 1 3368, 3988, 4608, 18359-18445 485 POUclass 5 448657 605 416165 1229 2129, 2749, homeobox 1 3369, 3989, 4609486 POU class 5 451077 606 391507 1230 2130, 2750, homeobox 1 3370,3990, 4610 487 POU class 5 454714 607 400047 1231 2131, 2751, homeobox 13371, 3991, 4611 488 POU class 5 546505 608 448154 1232 2132, 2752,homeobox 1 3372, 3992, 4612 489 POU class 5 547234 609 449442 1233 2133,2753, homeobox 1 3373, 3993, 4613 490 POU class 5 547658 610 446962 12342134, 2754, homeobox 1 3374, 3994, 4614 491 POU class 5 548682 611446815 1235 2135, 2755, homeobox 1 3375, 3995, 4615 492 POU class 5550059 612 447874 1236 2136, 2756, homeobox 1 3376, 3996, 4616 493 POUclass 5 550521 613 447969 1237 2137, 2757, homeobox 1 3377, 3997, 4617494 POU class 5 550572 614 448254 1238 2138, 2758, homeobox 1 3378,3998, 4618 495 POU class 5 553069 615 448231 1239 2139, 2759, homeobox 13379, 3999, 4619 496 POU class 5 553206 616 446757 1240 2140, 2760,homeobox 1 3380, 4000, 4620 497 proprotein 302118 617 303208 1241 2141,2761, convertase 3381, 4001, subtilisin/kexin 4621 type 9 498 proprotein452118 618 401598 1242 2142, 2762, convertase 3382, 4002,subtilisin/kexin 4622 type 9 499 proprotein 543384 619 441859 1243 2143,2763, convertase 3383, 4003, subtilisin/kexin 4623 type 9 500 protein C234071 620 234071 1244 2144, 2764, (inactivator of 3384, 4004,coagulation factors 4624 Va and VIIIa) 501 protein C 409048 621 3866791245 2145, 2765, (inactivator of 3385, 4005, coagulation factors 4625,5009, Va and VIIIa) 5339 502 protein C 422777 622 409543 1246 2146,2766, (inactivator of 3386, 4006, coagulation factors 4626, 5010, Va andVIIIa) 5340 503 protein C 427769 623 406295 1247 2147, 2767,(inactivator of 3387, 4007, coagulation factors 4627, 5011, Va andVIIIa) 5341 504 protein C 429925 624 412697 1248 2148, 2768,(inactivator of 3388, 4008, coagulation factors 4628, 5012, Va andVIIIa) 5342 505 protein C 453608 625 404030 1249 2149, 2769,(inactivator of 3389, 4009, coagulation factors 4629, 5013, Va andVIIIa) 5343 506 protein C 537436 626 442106 1250 2150, 2770,(inactivator of 3390, 4010, coagulation factors 4630, 5014, Va andVIIIa) 5344 507 relaxin 2 308420 627 308018 1251 2151, 2771, 3391, 4011,4631 508 relaxin 2 381627 628 371040 1252 2152, 2772, 3392, 4012, 4632,18446-18532 509 Reverse Caspase 3 1543 (cleavable) 510 Reverse Caspase 31544 (non-cleavable) 511 Reverse Caspase 6 1545 512 rhodopsin 296271 629296271 1253 2153, 2773, 3393, 4013, 4633, 5015, 5345, 18533-18619 513Rituximab; rituximab 514 serpin peptidase 355814 630 348068 1254 2154,2774, 1662 inhibitor, clade A 3394, 4014, (alpha-1 4634, 5016,antiproteinase, 5346, 18620-18706 antitrypsin), member 1 515 serpinpeptidase 393087 631 376802 1255 2155, 2775, 1662 inhibitor, clade A3395, 4015, (alpha-1 4635, 5017, antiproteinase, 5347 antitrypsin),member 1 516 serpin peptidase 393088 632 376803 1256 2156, 2776, 1662inhibitor, clade A 3396, 4016, (alpha-1 4636, 5018, antiproteinase, 5348antitrypsin), member 1 517 serpin peptidase 402629 633 386094 1257 2157,2777, inhibitor, clade A 3397, 4017, (alpha-1 4637, 5019,antiproteinase, 5349 antitrypsin), member 1 518 serpin peptidase 404814634 385960 1258 2158, 2778, 1662 inhibitor, clade A 3398, 4018, (alpha-14638, 5020, antiproteinase, 5350 antitrypsin), member 1 519 serpinpeptidase 437397 635 408474 1259 2159, 2779, 1662 inhibitor, clade A3399, 4019, (alpha-1 4639, 5021, antiproteinase, 5351 antitrypsin),member 1 520 serpin peptidase 440909 636 390299 1260 2160, 2780, 1662inhibitor, clade A 3400, 4020, (alpha-1 4640, 5022, antiproteinase, 5352antitrypsin), member 1 521 serpin peptidase 448921 637 416066 1261 2161,2781, 1662 inhibitor, clade A 3401, 4021, (alpha-1 4641, 5023,antiproteinase, 5353 antitrypsin), member 1 522 serpin peptidase 449399638 416354 1262 2162, 2782, 1662 inhibitor, clade A 3402, 4022, (alpha-14642, 5024, antiproteinase, 5354 antitrypsin), member 1 523 serpinpeptidase 553327 639 452480 1263 2163, 2783, inhibitor, clade A 3403,4023, (alpha-1 4643, 5025, antiproteinase, 5355, 18707-18793antitrypsin), member 1 524 serpin peptidase 556091 640 452169 1264 2164,2784, inhibitor, clade A 3404, 4024, (alpha-1 4644, 5026,antiproteinase, 5356 antitrypsin), member 1 525 serpin peptidase 556955641 451098 1265 2165, 2785, inhibitor, clade A 3405, 4025, (alpha-14645, 5027, antiproteinase, 5357 antitrypsin), member 1 526 serpinpeptidase 557118 642 451826 1266 2166, 2786, inhibitor, clade A 3406,4026, (alpha-1 4646, 5028, antiproteinase, 5358 antitrypsin), member 1527 serpin peptidase 557492 643 452452 1267 2167, 2787, inhibitor, cladeA 3407, 4027, (alpha-1 4647, 5029, antiproteinase, 5359 antitrypsin),member 1 528 serpin peptidase 351522 644 307953 1268 2168, 2788,inhibitor, clade C 3408, 4028, (antithrombin), 4648, 5030, member 1 5360529 serpin peptidase 367698 645 356671 1269 1546, 2169, inhibitor, cladeC 2789, 3409, (antithrombin), 4029, 4649, member 1 5031, 5361,18794-18932 530 serpin peptidase 324015 646 321853 1270 1547, 2170,inhibitor, clade F 2790, 3410, (alpha-2 4030, 4650 antiplasmin, pigmentepithelium derived factor), member 2 531 serpin peptidase 382061 647371493 1271 2171, 2791, inhibitor, clade F 3411, 4031, (alpha-2 4651,18933-19071 antiplasmin, pigment epithelium derived factor), member 2532 serpin peptidase 450523 648 403877 1272 1548, 2172, inhibitor, cladeF 2792, 3412, (alpha-2 4032, 4652 antiplasmin, pigment epitheliumderived factor), member 2 533 serpin peptidase 453066 649 402286 12731549, 2173, inhibitor, clade F 2793, 3413, (alpha-2 4033, 4653antiplasmin, pigment epithelium derived factor), member 2 534 serpinpeptidase 278407 650 278407 1274 1550, 2174, inhibitor, clade G 2794,3414, (C1 inhibitor), 4034, 4654, member 1 5032, 5362, 19072-19210 535serpin peptidase 378323 651 367574 1275 1551, 2175, inhibitor, clade G2795, 3415, (C1 inhibitor), 4035, 4655, member 1 5033, 5363 536 serpinpeptidase 378324 652 367575 1276 1552, 2176, inhibitor, clade G 2796,3416, (C1 inhibitor), 4036, 4656, member 1 5034, 5364 537 serpinpeptidase 405496 653 384561 1277 1553, 2177, inhibitor, clade G 2797,3417, (C1 inhibitor), 4037, 4657, member 1 5035, 5365 538 serpinpeptidase 433668 654 399800 1278 2178, 2798, inhibitor, clade G 3418,4038, (C1 inhibitor), 4658, 5036, member 1 5366 539 sirtuin 1 212015 655212015 1279 1554, 2179, 1663 2799, 3419, 4039, 4659, 19211-19297 540sirtuin 1 403579 656 384063 1280 1555, 2180, 2800, 3420, 4040, 4660,5037, 5367 541 sirtuin 1 406900 657 384508 1281 2181, 2801, 3421, 4041,4661, 5038, 5368 542 sirtuin 1 432464 658 409208 1282 1556, 2182, 16642802, 3422, 4042, 4662, 5039, 5369 543 sirtuin 6 305232 659 305310 12831557, 2183, 2803, 3423, 4043, 4663, 5040, 5370 544 sirtuin 6 337491 660337332 1284 1558, 2184, 1665 2804, 3424, 4044, 4664, 5041, 5371,19298-19384 545 sirtuin 6 381935 661 371360 1285 1559, 2185, 2805, 3425,4045, 4665, 5042, 5372 546 solute carrier 314251 662 323568 1286 1560,2186, family 2 2806, 3426, (facilitated glucose 4046, 4666,transporter), 19385-19523 member 2 547 solute carrier 382808 663 3722581287 1561, 2187, family 2 2807, 3427, (facilitated glucose 4047, 4667transporter), member 2 548 solute carrier 577093 664 461344 1288 2188,2808, family 37 3428, 4048, (glucose-6- 4668,, 19524-19610 phosphatetransporter) member 4 549 solute carrier 590663 665 464769 1289 2189,2809, family 37 3429, 4049, (glucose-6- 4669 phosphate transporter)member 4 550 solute carrier 261024 666 261024 1290 2190, 2810, family 40(iron- 3430, 4050, regulated 4670 transporter), member 1 551 solutecarrier 427241 667 390005 1291 2191, 2811, family 40 (iron- 3431, 4051,regulated 4671 transporter), member 1 552 solute carrier 427419 668392730 1292 2192, 2812, family 40 (iron- 3432, 4052, regulated 4672transporter), member 1 553 solute carrier 440626 669 396134 1293 2193,2813, family 40 (iron- 3433, 4053, regulated 4673 transporter), member 1554 solute carrier 455320 670 413549 1294 2194, 2814, family 40 (iron-3434, 4054, regulated 4674 transporter), member 1 555 solute carrier481497 671 446069 1295 2195, 2815, family 40 (iron- 3435, 4055,regulated 4675 transporter), member 1 556 solute carrier 544056 672444582 1296 2196, 2816, family 40 (iron- 3436, 4056, regulated 4676transporter), member 1 557 sortilin 1 256637 673 256637 1297 1562, 2197,2817, 3437, 4057, 4677, 5043, 5373 558 sortilin 1 538502 674 438597 12981563, 2198, 2818, 3438, 4058, 4678, 5044, 5374, 19611-19697 559 spermadhesion 223028 675 223028 1299 2199, 2819, molecule 1 (PH-20 3439,4059, hyaluronidase, 4679 zona pellucida binding) 560 sperm adhesion340011 676 345849 1300 2200, 2820, molecule 1 (PH-20 3440, 4060,hyaluronidase, 4680, 5045, zona pellucida 5375 binding) 561 spermadhesion 402183 677 386028 1301 2201, 2821, molecule 1 (PH-20 3441,4061, hyaluronidase, 4681, 5046, zona pellucida 5376 binding) 562 spermadhesion 413927 678 391491 1302 2202, 2822, molecule 1 (PH-20 3442,4062, hyaluronidase, 4682, 5047, zona pellucida 5377 binding) 563 spermadhesion 439500 679 402123 1303 2203, 2823, molecule 1 (PH-20 3443,4063, hyaluronidase, 4683, 5048, zona pellucida 5378 binding) 564 spermadhesion 460182 680 417934 1304 2204, 2824, molecule 1 (PH-20 3444,4064, hyaluronidase, 4684, 5049, zona pellucida 5379 binding) 565sphingomyelin 299397 681 299397 1305 1564, 2205, phosphodiesterase 2825,3445, 1, acid lysosomal 4065, 4685, 5050, 5380, 566 sphingomyelin 342245682 340409 1306 1565, 2206, 1666 phosphodiesterase 2826, 3446, 1, acidlysosomal 4066, 4686, 5051, 5381, 19698-19784 567 sphingomyelin 356761683 349203 1307 1566, 2207, phosphodiesterase 2827, 3447, 1, acidlysosomal 4067, 4687, 5052, 5382 568 sphingomyelin 527275 684 4353501308 1567, 2208, phosphodiesterase 2828, 3448, 1, acid lysosomal 4068,4688, 5053, 5383 569 sphingomyelin 533123 685 435950 1309 1568, 2209,phosphodiesterase 2829, 3449, 1, acid lysosomal 4069, 4689, 5054, 5384570 sphingomyelin 534405 686 434353 1310 1569, 2210, phosphodiesterase2830, 3450, 1, acid lysosomal 4070, 4690, 5055, 5385 571 SRY (sex 325404687 323588 1311 2211, 2831, determining region 3451, 4071, Y)-box 2 4691572 SRY (sex 431565 688 439111 1312 2212, 2832, determining region 3452,4072, Y)-box 2 4692, 19785-19871 573 Subcutaneous Herceptin; trastuzumab574 surfactant protein B 393822 689 377409 1313 2213, 2833, 3453, 4073,4693 575 surfactant protein B 409383 690 386346 1314 2214, 2834, 3454,4074, 4694 576 surfactant protein B 441838 691 395757 1315 2215, 2835,3455, 4075, 4695 577 surfactant protein B 519937 692 428719 1316 2216,2836, 3456, 4076, 4696 578 Synagis 579 thrombomodulin 377103 693 3663071317 1570, 2217, 2837, 3457, 4077, 4697, 19872-19958 580 thrombomodulin503590 694 440119 1318 2218, 2838, 3458, 4078, 4698 581 thrombopoietin204615 695 204615 1319 1571, 2219, 1667 2839, 3459, 4079, 4699, 5056,5386, 19959-20045 582 thrombopoietin 353488 696 341335 1320 2220, 2840,3460, 4080, 4700, 5057, 5387 583 thrombopoietin 421442 697 411704 13211572, 2221, 2841, 3461, 4081, 4701, 5058, 5388 584 thrombopoietin 445696698 410763 1322 1573, 2222, 2842, 3462, 4082, 4702, 5059, 5389 585TL011; rituximab 586 transferrin 223051 699 223051 1323 2223, 2843,receptor 2 3463, 4083, 4703 587 transferrin 431692 700 413905 1324 2224,2844, receptor 2 3464, 4084, 4704 588 transferrin 462107 701 420525 13252225, 2845, receptor 2 3465, 4085, 4705 589 transferrin 544242 702443656 1326 2226, 2846, receptor 2 3466, 4086, 4706 590 transforming221930 703 221930 1327 2227, 2847, 1668 growth factor, beta 1 3467,4087, 4707, 20046-20132 591 transforming 366929 704 355896 1328 2228,2848, growth factor, beta 2 3468, 4088, 4708 592 transforming 366930 705355897 1329 2229, 2849, growth factor, beta 2 3469, 4089, 4709 593transforming 238682 706 238682 1330 2230, 2850, growth factor, beta 33470, 4090, 4710, 5060, 5390, 20133-20219 594 transforming 556285 707451110 1331 2231, 2851, growth factor, beta 3 3471, 4091, 4711, 5061,5391 595 transthyretin 237014 708 237014 1332 2232, 2852, 3472, 4092,4712, 20220-20358 596 transthyretin 432547 709 396762 1333 2233, 2853,3473, 4093, 4713 597 transthyretin 541025 710 442134 1334 2234, 2854,3474, 4094, 4714 598 tuftelin 1 353024 711 343781 1335 2235, 2855, 16693475, 4095, 4715, 5062, 5392 599 tuftelin 1 368848 712 357841 1336 2236,2856, 1669 3476, 4096, 4716, 5063, 5393 600 tuftelin 1 368849 713 3578421337 2237, 2857, 1669 3477, 4097, 4717, 5064, 5394, 20359-20445 601tuftelin 1 507671 714 443643 1338 2238, 2858, 1669 3478, 4098, 4718,5065, 5395 602 tuftelin 1 538902 715 437997 1339 2239, 2859, 3479, 4099,4719, 5066, 5396 603 tuftelin 1 544350 716 441557 1340 2240, 2860, 16693480, 4100, 4720, 5067, 5397 604 tumor protein p53 269305 717 2693051341 1574, 2241, 1670 2861, 3481, 4101, 4721, 5068, 5398, 20446-20532605 tumor protein p53 359597 718 352610 1342 1575, 2242, 2862, 3482,4102, 4722, 5069, 5399 606 tumor protein p53 396473 719 379735 13432243, 2863, 3483, 4103, 4723, 5070, 5400 607 tumor protein p53 413465720 410739 1344 1576, 2244, 2864, 3484, 4104, 4724, 5071, 5401 608 tumorprotein p53 414315 721 394195 1345 2245, 2865, 3485, 4105, 4725, 5072,5402 609 tumor protein p53 419024 722 402130 1346 2246, 2866, 3486,4106, 4726, 5073, 5403 610 tumor protein p53 420246 723 391127 13471577, 2247, 2867, 3487, 4107, 4727, 5074, 5404 611 tumor protein p53445888 724 391478 1348 2248, 2868, 1670 3488, 4108, 4728, 5075, 5405 612tumor protein p53 455263 725 398846 1349 1578, 2249, 2869, 3489, 4109,4729, 5076, 5406 613 tumor protein p53 503591 726 426252 1350 1579,2250, 2870, 3490, 4110, 4730, 5077, 5407 614 tumor protein p53 508793727 424104 1351 1580, 2251, 2871, 3491, 4111, 4731, 5078, 5408 615 tumorprotein p53 509690 728 425104 1352 1581,, 2252, 2872, 3492, 4112, 4732,5079, 5409 616 tumor protein p53 514944 729 423862 1353 1582, 2253,2873, 3493, 4113, 4733, 5080, 5410 617 tumor protein p53 545858 730437792 1354 2254, 2874, 3494, 4114, 4734, 5081, 5411 618 tyrosinase263321 731 263321 1355 2255, 2875, (oculocutaneous 3495, 4115, albinismIA) 4735,,, 20533-20619 619 tyrosine 355962 732 348234 1356 1583, 2256,aminotransferase 2876, 3496, 4116, 4736,,, 20620-20758 620 UDP 305208733 304845 1357 1584, 2257, glucuronosyltransferase 2877, 3497, 1family, 4117, 4737,,, polypeptide A1 20759-20845 621 UDP 360418 734353593 1358 1585, 2258, glucuronosyltransferase 2878, 3498, 1 family,4118, 4738 polypeptide A1 622 UDP 344644 735 343838 1359 2259, 2879,glucuronosyltransferase 3499, 4119, 1 family, 4739 polypeptide A10 623UDP 482026 736 418532 1360 2260, 2880, glucuronosyltransferase 3500,4120, 1 family, 4740 polypeptide A3 624 UDP 513300 737 427404 1361 2261,2881, glycosyltransferase 3501, 4121, 3 family 4741, 5082, polypeptideA2 5412 625 UDP 282507 738 282507 1362 2262, 2882, 1671glycosyltransferase 3502, 4122, 3 family, 4742, 5083, polypeptide A25413 626 UDP 515131 739 420865 1363 2263, 2883, glycosyltransferase3503, 4123, 3 family, 4743, 5084, polypeptide A2 5414 627 UDP 545528 740445367 1364 2264, 2884, glycosyltransferase 3504, 4124, 3 family, 4744,5085, polypeptide A2 5415 628 vascular 230480 741 230480 1365 1586,2265, endothelial growth 2885, 3505, factor A 4125, 4745 629 vascular324450 742 317598 1366 1587, 2266, endothelial growth 2886, 3506, factorA 4126, 4746, 5086, 5416 630 vascular 372055 743 361125 1367 1588, 2267,endothelial growth 2887, 3507, factor A 4127, 4747, 5087, 5417 631vascular 372064 744 361134 1368 1589, 2268, endothelial growth 2888,3508, factor A 4128, 4748, 5088, 5418 632 vascular 372067 745 3611371369 1590, 2269, 1672 endothelial growth 2889, 3509, factor A 4129,4749, 5089, 5419, 20846-20932 633 vascular 372077 746 361148 1370 1591,2270, endothelial growth 2890, 3510, factor A 4130, 4750, 5090, 5420 634vascular 413642 747 389864 1371 1592, 2271, endothelial growth 2891,3511, factor A 4131, 4751, 5091, 5421 635 vascular 417285 748 3886631372 1593, 2272, endothelial growth 2892, 3512, factor A 4132, 4752,5092, 5422 636 vascular 425836 749 388465 1373 1594, 2273, endothelialgrowth 2893, 3513, factor A 4133, 4753, 5093, 5423 637 vascular 457104750 409911 1374 1595, 2274, endothelial growth 2894, 3514, factor A4134, 4754, 5094, 5424 638 vascular 482630 751 421561 1375 1596, 2275,endothelial growth 2895, 3515, factor A 4135, 4755, 5095, 5425 639vascular 518689 752 430829 1376 1597, 2276, endothelial growth 2896,3516, factor A 4136, 4756, 5096, 5426 640 vascular 518824 753 4300021377 1598, 2277, endothelial growth 2897, 3517, factor A 4137, 4757,5097, 5427 641 vascular 519767 754 430594 1378 1599, 2278, endothelialgrowth 2898, 3518, factor A 4138, 4758, 5098, 5428 642 vascular 520948755 428321 1379 1600, 2279, endothelial growth 2899, 3519, factor A4139, 4759, 5099, 5429 643 vascular 523125 756 429008 1380 1601, 2280,endothelial growth 2900, 3520, factor A 4140, 4760, 5100, 5430 644vascular 523873 757 430479 1381 1602, 2281, endothelial growth 2901,3521, factor A 4141, 4761, 5101, 5431 645 vascular 523950 758 4296431382 1603, 2282, endothelial growth 2902, 3522, factor A 4142, 4762,5102, 5432 646 vascular 280193 759 280193 1383 1604, 2283, endothelialgrowth 2903, 3523, factor C 4143, 4763, 20933-21019 647 vasoactive367243 760 356212 1384 1605, 2284, intestinal peptide 2904, 3524, 4144,4764 648 vasoactive 367244 761 356213 1385 1606, 2285, intestinalpeptide 2905, 3525, 4145, 4765, 21020-21106 649 vasoactive 431366 762410356 1386 1607, 2286, intestinal peptide 2906, 3526, 4146, 4766 650v-myc 259523 763 259523 1387 2287, 2907, myelocytomatosis 3527, 4147,viral oncogene 4767 homolog (avian) 651 v-myc 377970 764 367207 13882288, 2908, myelocytomatosis 3528, 4148, viral oncogene 4768,21107-21110 homolog (avian) 652 v-myc 454617 765 405312 1389 2289, 2909,myelocytomatosis 3529, 4149, viral oncogene 4769 homolog (avian) 653v-myc 524013 766 430235 1390 2290, 2910, myelocytomatosis 3530, 4150,viral oncogene 4770, 21111-21284 homolog (avian) 654 von Willebrand261405 767 261405 1391 1608, 2291, factor 2911, 3531, 4151, 4771,21285-21423 655 zinc finger, 403903 768 384434 1392 2292, 2912,GATA-like 3532, 4152, protein 1 4772

Protein Cleavage Signals and Sites

In one embodiment, the polypeptides of the present invention may includeat least one protein cleavage signal containing at least one proteincleavage site. The protein cleavage site may be located at theN-terminus, the C-terminus, at any space between the N- and theC-termini such as, but not limited to, half-way between the N- andC-termini, between the N-terminus and the half way point, between thehalf way point and the C-terminus, and combinations thereof.

The polypeptides of the present invention may include, but is notlimited to, a proprotein convertase (or prohormone convertase), thrombinor Factor Xa protein cleavage signal. Proprotein convertases are afamily of nine proteinases, comprising seven basic amino acid-specificsubtilisin-like serine proteinases related to yeast kexin, known asprohormone convertase 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basicamino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilasesthat cleave at non-basic residues, called subtilisin kexin isozyme 1(SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9).Non-limiting examples of protein cleavage signal amino acid sequencesare listing in Table 7. In Table 7, “X” refers to any amino acid, “n”may be 0, 2, 4 or 6 amino acids and “*” refers to the protein cleavagesite. In Table 7, SEQ ID NO: 21426 refers to when n=4 and SEQ ID NO:21427 refers to when n=6.

TABLE 7 Protein Cleavage Site Sequences Protein Cleavage Signal AminoAcid Cleavage Sequence SEQ ID NO Proprotein R-X-X-R* 21424 convertaseR-X-K/R-R* 21425 K/R-Xn-K/R* 21426 or 21427 Thrombin L-V-P-R*-G-S 21428L-V-P-R* 21429 A/F/G/I/L/T/V/M-A/F/G/I/L/T/ 21430 V/W-P-R* Factor XaI-E-G-R* 21431 I-D-G-R* 21432 A-E-G-R* 21433 A/F/G/I/L/T/V/M-D/E-G-R*21434

In one embodiment, the primary constructs and the mmRNA of the presentinvention may be engineered such that the primary construct or mmRNAcontains at least one encoded protein cleavage signal. The encodedprotein cleavage signal may be located before the start codon, after thestart codon, before the coding region, within the coding region such as,but not limited to, half way in the coding region, between the startcodon and the half way point, between the half way point and the stopcodon, after the coding region, before the stop codon, between two stopcodons, after the stop codon and combinations thereof.

In one embodiment, the primary constructs or mmRNA of the presentinvention may include at least one encoded protein cleavage signalcontaining at least one protein cleavage site. The encoded proteincleavage signal may include, but is not limited to, a proproteinconvertase (or prohormone convertase), thrombin and/or Factor Xa proteincleavage signal. One of skill in the art may use Table 1 above or otherknown methods to determine the appropriate encoded protein cleavagesignal to include in the primary constructs or mmRNA of the presentinvention. For example, starting with the signal of Table 7 andconsidering the codons of Table 1 one can design a signal for theprimary construct which can produce a protein signal in the resultingpolypeptide.

In one embodiment, the polypeptides of the present invention include atleast one protein cleavage signal and/or site.

As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S. Pub. No.20090227660, herein incorporated by reference in their entireties, use afurin cleavage site to cleave the N-terminal methionine of GLP-1 in theexpression product from the Golgi apparatus of the cells. In oneembodiment, the polypeptides of the present invention include at leastone protein cleavage signal and/or site with the proviso that thepolypeptide is not GLP-1.

In one embodiment, the primary constructs or mmRNA of the presentinvention includes at least one encoded protein cleavage signal and/orsite.

In one embodiment, the primary constructs or mmRNA of the presentinvention includes at least one encoded protein cleavage signal and/orsite with the proviso that the primary construct or mmRNA does notencode GLP-1.

In one embodiment, the primary constructs or mmRNA of the presentinvention may include more than one coding region. Where multiple codingregions are present in the primary construct or mmRNA of the presentinvention, the multiple coding regions may be separated by encodedprotein cleavage sites. As a non-limiting example, the primary constructor mmRNA may be signed in an ordered pattern. On such pattern followsAXBY form where A and B are coding regions which may be the same ordifferent coding regions and/or may encode the same or differentpolypeptides, and X and Y are encoded protein cleavage signals which mayencode the same or different protein cleavage signals. A second suchpattern follows the form AXYBZ where A and B are coding regions whichmay be the same or different coding regions and/or may encode the sameor different polypeptides, and X, Y and Z are encoded protein cleavagesignals which may encode the same or different protein cleavage signals.A third pattern follows the form ABXCY where A, B and C are codingregions which may be the same or different coding regions and/or mayencode the same or different polypeptides, and X and Y are encodedprotein cleavage signals which may encode the same or different proteincleavage signals.

In one embodiment, the polypeptides, primary constructs and mmRNA canalso contain sequences that encode protein cleavage sites so that thepolypeptides, primary constructs and mmRNA can be released from acarrier region or a fusion partner by treatment with a specific proteasefor said protein cleavage site.

In one embodiment, the polypeptides, primary constructs and mmRNA of thepresent invention may include a sequence encoding the 2A peptide. In oneembodiment, this sequence may be used to separate the coding region oftwo or more polypeptides of interest. As a non-limiting example, thesequence encoding the 2A peptide may be between coding region A andcoding region B (A-2Apep-B). The presence of the 2A peptide would resultin the cleavage of one long protein into protein A, protein B and the 2Apeptide. Protein A and protein B may be the same or differentpolypeptides of interest. In another embodiment, the 2A peptide may beused in the polynucleotides, primary constructs and/or mmRNA of thepresent invention to produce two, three, four, five, six, seven, eight,nine, ten or more proteins.

Incorporating Post Transcriptional Control Modulators

In one embodiment, the polynucleotides, primary constructs and/or mmRNAof the present invention may include at least one post transcriptionalcontrol modulator. These post transcriptional control modulators may be,but are not limited to, small molecules, compounds and regulatorysequences. As a non-limiting example, post transcriptional control maybe achieved using small molecules identified by PTC Therapeutics Inc.(South Plainfield, N.J.) using their GEMS™ (Gene Expression Modulationby Small-Moleclues) screening technology.

The post transcriptional control modulator may be a gene expressionmodulator which is screened by the method detailed in or a geneexpression modulator described in International Publication No.WO2006022712, herein incorporated by reference in its entirety. Methodsidentifying RNA regulatory sequences involved in translational controlare described in International Publication No. WO2004067728, hereinincorporated by reference in its entirety; methods identifying compoundsthat modulate untranslated region dependent expression of a gene aredescribed in International Publication No. WO2004065561, hereinincorporated by reference in its entirety.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAof the present invention may include at least one post transcriptionalcontrol modulator is located in the 5′ and/or the 3′ untranslated regionof the polynucleotides, primary constructs and/or mmRNA of the presentinvention

In another embodiment, the polynucleotides, primary constructs and/ormmRNA of the present invention may include at least one posttranscription control modulator to modulate premature translationtermination. The post transcription control modulators may be compoundsdescribed in or a compound found by methods outlined in InternationalPublication Nso. WO2004010106, WO2006044456, WO2006044682, WO2006044503and WO2006044505, each of which is herein incorporated by reference inits entirety. As a non-limiting example, the compound may bind to aregion of the 28S ribosomal RNA in order to modulate prematuretranslation termination (See e.g., WO2004010106, herein incorporated byreference in its entirety).

In one embodiment, polynucleotides, primary constructs and/or mmRNA ofthe present invention may include at least one post transcriptioncontrol modulator to alter protein expression. As a non-limitingexample, the expression of VEGF may be regulated using the compoundsdescribed in or a compound found by the methods described inInternational Publication Nos. WO2005118857, WO2006065480, WO2006065479and WO2006058088, each of which is herein incorporated by reference inits entirety.

The polynucleotides, primary constructs and/or mmRNA of the presentinvention may include at least one post transcription control modulatorto control translation. In one embodiment, the post transcriptioncontrol modulator may be a RNA regulatory sequence. As a non-limitingexample, the RNA regulatory sequence may be identified by the methodsdescribed in International Publication No. WO2006071903, hereinincorporated by reference in its entirety.

III. Modifications

Herein, in a polynucleotide (such as a primary construct or an mRNAmolecule), the terms “modification” or, as appropriate, “modified” referto modification with respect to A, G, U or C ribonucleotides. Generally,herein, these terms are not intended to refer to the ribonucleotidemodifications in naturally occurring 5′-terminal mRNA cap moieties. In apolypeptide, the term “modification” refers to a modification ascompared to the canonical set of 20 amino acids, moiety)

The modifications may be various distinct modifications. In someembodiments, the coding region, the flanking regions and/or the terminalregions may contain one, two, or more (optionally different) nucleosideor nucleotide modifications.

In some embodiments, a modified polynucleotide, primary construct, ormmRNA introduced to a cell may exhibit reduced degradation in the cell,as compared to an unmodified polynucleotide, primary construct, ormmRNA.

The polynucleotides, primary constructs, and mmRNA can include anyuseful modification, such as to the sugar, the nucleobase, or theinternucleoside linkage (e.g. to a linking phosphate/to a phosphodiesterlinkage/to the phosphodiester backbone). One or more atoms of apyrimidine nucleobase may be replaced or substituted with optionallysubstituted amino, optionally substituted thiol, optionally substitutedalkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). Incertain embodiments, modifications (e.g., one or more modifications) arepresent in each of the sugar and the internucleoside linkage.Modifications according to the present invention may be modifications ofribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threosenucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids(PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additionalmodifications are described herein.

As described herein, the polynucleotides, primary constructs, and mmRNAof the invention do not substantially induce an innate immune responseof a cell into which the mRNA is introduced. Featues of an inducedinnate immune response include 1) increased expression ofpro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I,MDA5, etc, and/or 3) termination or reduction in protein translation.

In certain embodiments, it may desirable to intracellularly degrade amodified nucleic acid molecule introduced into the cell. For example,degradation of a modified nucleic acid molecule may be preferable ifprecise timing of protein production is desired. Thus, in someembodiments, the invention provides a modified nucleic acid moleculecontaining a degradation domain, which is capable of being acted on in adirected manner within a cell. In another aspect, the present disclosureprovides polynucleotides comprising a nucleoside or nucleotide that candisrupt the binding of a major groove interacting, e.g. binding, partnerwith the polynucleotide (e.g., where the modified nucleotide hasdecreased binding affinity to major groove interacting partner, ascompared to an unmodified nucleotide).

The polynucleotides, primary constructs, and mmRNA can optionallyinclude other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs,shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAsthat induce triple helix formation, aptamers, vectors, etc.). In someembodiments, the polynucleotides, primary constructs, or mmRNA mayinclude one or more messenger RNAs (mRNAs) and one or more modifiednucleoside or nucleotides (e.g., mmRNA molecules). Details for thesepolynucleotides, primary constructs, and mmRNA follow.

Polynucleotides and Primary Constructs

The polynucleotides, primary constructs, and mmRNA of the inventionincludes a first region of linked nucleosides encoding a polypeptide ofinterest, a first flanking region located at the 5′ terminus of thefirst region, and a second flanking region located at the 3′ terminus ofthe first region.

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having Formula (Ia) orFormula (Ia-1):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

- - - is a single bond or absent;

each of R^(1′), R^(2′), R^(1″), R², R², R³, R⁴, and R⁵ is independently,if present, H, halo, hydroxy, thiol, optionally substituted alkyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, orabsent; wherein the combination of R³ with one or more of R1′, R1″, R2′,R2″, or R5 (e.g., the combination of R1′ and R3, the combination of R1″and R3, the combination of R2′ and R3, the combination of R2″ and R3, orthe combination of R5 and R3) can join together to form optionallysubstituted alkylene or optionally substituted heteroalkylene and, takentogether with the carbons to which they are attached, provide anoptionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl); wherein the combination of R5 with one ormore of R1′, R1″, R2′, or R2″ (e.g., the combination of R1′ and R5, thecombination of R1″ and R5, the combination of R2′ and R⁵, or thecombination of R²″ and R⁵) can join together to form optionallysubstituted alkylene or optionally substituted heteroalkylene and, takentogether with the carbons to which they are attached, provide anoptionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl); and wherein the combination of R⁴ and one ormore of R^(1′), R^(1″), R^(2′), R^(2″), R³, or R⁵ can join together toform optionally substituted alkylene or optionally substitutedheteroalkylene and, taken together with the carbons to which they areattached, provide an optionally substituted heterocyclyl (e.g., abicyclic, tricyclic, or tetracyclic heterocyclyl);each of m′ and m″ is,independently, an integer from 0 to 3 (e.g., from 0 to 2, from 0 to 1,from 1 to 3, or from 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or absent;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof), wherein the combination of B and R^(1′), the combination of Band R^(2′), the combination of B and R^(1″), or the combination of B andR^(2″) can, taken together with the carbons to which they are attached,optionally form a bicyclic group (e.g., a bicyclic heterocyclyl) orwherein the combination of B, R^(1″), and R³ or the combination of B,R^(2″), and R³ can optionally form a tricyclic or tetracyclic group(e.g., a tricyclic or tetracyclic heterocyclyl, such as in Formula(IIo)-(IIp) herein). In some embodiments, the polynucleotide, primaryconstruct, or mmRNA includes a modified ribose. In some embodiments, thepolynucleotide, primary construct, or mmRNA (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides having Formula (Ia-2)-(Ia-5) or a pharmaceuticallyacceptable salt or stereoisomer thereof

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, the first flanking region, or the secondflanking region) includes n number of linked nucleosides having Formula(Ib) or Formula (Ib-1):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

- - - is a single bond or absent;

each of R¹, R^(3′), R^(3″), and R⁴ is, independently, H, halo, hydroxy,optionally substituted alkyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionallysubstituted hydroxyalkoxy, optionally substituted amino, azido,optionally substituted aryl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, or absent; and wherein the combination of R¹ and R^(3′) orthe combination of R¹ and R^(3″) can be taken together to formoptionally substituted alkylene or optionally substituted heteroalkylene(e.g., to produce a locked nucleic acid);

each R⁵ is, independently, H, halo, hydroxy, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, or absent;

each of Y¹, Y², and Y³ is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted alkoxyalkoxy, or optionally substituted amino;

n is an integer from 1 to 100,000; and

B is a nucleobase.

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having Formula (Ic):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

- - - is a single bond or absent;

each of B¹, B², and B³ is, independently, a nucleobase (e.g., a purine,a pyrimidine, or derivatives thereof, as described herein), H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl, wherein one and only one of B¹, B²,and B³ is a nucleobase;

each of R^(b1), R^(b2),R^(b3), R³, and R⁵ is, independently, H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl oroptionally substituted aminoalkynyl;

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

wherein the ring including U can include one or more double bonds.

In particular embodiments, the ring including U does not have a doublebond between U-CB³R^(b3) or between CB³R^(b3)—C^(B2)R^(b2).

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having Formula (Id):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

each R³ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkynyl;

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, optionally substituted alkylene (e.g.,methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having Formula (Ie):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of U′ and U″ is, independently, O, S, N(R^(U))_(nu), orC(R^(U))_(nu), wherein nu is an integer from 0 to 2 and each R^(U) is,independently, H, halo, or optionally substituted alkyl;

each R⁶ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkynyl;

each Y^(5′) is, independently, O, S, optionally substituted alkylene(e.g., methylene or ethylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having Formula (If) or(If-1):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of U′ and U″ is, independently, O, S, N,N(R^(U))_(nu), orC(R^(U))_(nu), wherein nu is an integer from 0 to 2 and each R^(U) is,independently, H, halo, or optionally substituted alkyl (e.g., U′ is 0and U″ is N);

- - - is a single bond or absent;

each of R^(1′), R^(2′), R^(1″), R^(2″), R³, and R⁴ is, independently, H,halo, hydroxy, thiol, optionally substituted alkyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted aminoalkoxy, optionallysubstituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,optionally substituted amino, azido, optionally substituted aryl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, or absent; and wherein thecombination of R^(1′) and R³, the combination of R^(1″) and R³, thecombination of R^(2′) and R³, or the combination of R^(2″) and R³ can betaken together to form optionally substituted alkylene or optionallysubstituted heteroalkylene (e.g., to produce a locked nucleic acid);eachof m′ and m″ is, independently, an integer from 0 to 3 (e.g., from 0 to2, from 0 to 1, from 1 to 3, or from 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or absent;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If), and (IIa)-(IIp)), thering including U has one or two double bonds.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachof R¹, R^(1′), and R^(1″), if present, is H. In further embodiments,each of R², R^(2′), and R^(2″), if present, is, independently, H, halo(e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In particularembodiments, alkoxyalkoxy is —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl). In some embodiments, s2 is 0, s1 is 1 or 2,s3 is 0 or 1, and R′ is C₁₋₆ alkyl.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachof R², R^(2′), and R^(2″), if present, is H. In further embodiments,each of R¹, R^(1′), and R^(1″), if present, is, independently, H, halo(e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In particularembodiments, alkoxyalkoxy is —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl). In some embodiments, s2 is 0, s1 is 1 or 2,s3 is 0 or 1, and R′ is C₁₋₆ alkyl.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachof R³, R⁴, and R⁵ is, independently, H, halo (e.g., fluoro), hydroxy,optionally substituted alkyl, optionally substituted alkoxy (e.g.,methoxy or ethoxy), or optionally substituted alkoxyalkoxy. Inparticular embodiments, R³ is H, R⁴ is H, R⁵ is H, or R³, R⁴, and R⁵ areall H. In particular embodiments, R³ is C₁₋₆ alkyl, R⁴ is C₁₋₆ alkyl, R⁵is C₁₋₆ alkyl, or R³, R⁴, and R⁵ are all C₁₋₆ alkyl. In particularembodiments, R³ and R⁴ are both H, and R⁵ is C₁₋₆ alkyl.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R³ andR⁵ join together to form optionally substituted alkylene or optionallysubstituted heteroalkylene and, taken together with the carbons to whichthey are attached, provide an optionally substituted heterocyclyl (e.g.,a bicyclic, tricyclic, or tetracyclic heterocyclyl, such as trans-3′,4′analogs, wherein R³ and R⁵ join together to form heteroalkylene (e.g.,—(CH₂)_(b1)—O—(CH₂)_(b2)—O—(CH₂)_(b3)—, wherein each of b1, b2, and b3are, independently, an integer from 0 to 3).

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R³ andone or more of R^(1′), R^(1″), R^(2′), R^(2″), or R⁵ join together toform optionally substituted alkylene or optionally substitutedheteroalkylene and, taken together with the carbons to which they areattached, provide an optionally substituted heterocyclyl (e.g., abicyclic, tricyclic, or tetracyclic heterocyclyl, R³ and one or more ofR^(1′), R^(1″), R^(2′), R^(2″), or R⁵ join together to formheteroalkylene (e.g., —(CH₂)_(b1)—O—(CH₂)_(b2)—O—(CH₂)_(b3)—, whereineach of b1, b2, and b3 are, independently, an integer from 0 to 3).

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R⁵ andone or more of R^(1′), R^(1″), R^(2′), or R^(2″) join together to formoptionally substituted alkylene or optionally substituted heteroalkyleneand, taken together with the carbons to which they are attached, providean optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl, R⁵ and one or more of R^(1′), R^(1″), R^(2′)or R^(2″) join together to form heteroalkylene (e.g.,—(CH₂)_(b1)—O—(CH₂)_(b2)—O—(CH₂)_(b3)—, wherein each of b1, b2, and b3are, independently, an integer from 0 to 3).

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachY² is, independently, O, S, or —NR^(N1)—, wherein R^(N1) is H,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl. In particularembodiments, Y² is NR^(N1)—, wherein R^(N1) is H or optionallysubstituted alkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl,or n-propyl).

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachY³ is, independently, O or S.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R¹ isH; each R² is, independently, H, halo (e.g., fluoro), hydroxy,optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionallysubstituted alkoxyalkoxy (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is0 or 1, and R′ is C₁₋₆ alkyl); each Y² is, independently, O or—NR^(N1)—, wherein R^(N1) is H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, or optionallysubstituted aryl (e.g., wherein R^(N1) is H or optionally substitutedalkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl, orn-propyl)); and each Y³ is, independently, O or S (e.g., S). In furtherembodiments, R³ is H, halo (e.g., fluoro), hydroxy, optionallysubstituted alkyl, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In yet furtherembodiments, each Y¹ is, independently, O or —NR^(N1)—, wherein R^(N1)is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, or optionally substituted aryl (e.g.,wherein R^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆ alkyl,such as methyl, ethyl, isopropyl, or n-propyl)); and each Y⁴ is,independently, H, hydroxy, thiol, optionally substituted alkyl,optionally substituted alkoxy, optionally substituted thioalkoxy,optionally substituted alkoxyalkoxy, or optionally substituted amino.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachR¹ is, independently, H, halo (e.g., fluoro), hydroxy, optionallysubstituted alkoxy (e.g., methoxy or ethoxy), or optionally substitutedalkoxyalkoxy (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each ofs2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H orC₁₋₂₀ alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′is C₁₋₆ alkyl); R² is H; each Y² is, independently, O or —NR^(N1)—,wherein R^(N1) is H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, or optionallysubstituted aryl (e.g., wherein R^(N1) is H or optionally substitutedalkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl, orn-propyl)); and each Y³ is, independently, O or S (e.g., S). In furtherembodiments, R³ is H, halo (e.g., fluoro), hydroxy, optionallysubstituted alkyl, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In yet furtherembodiments, each Y¹ is, independently, O or —NR^(N1)—, wherein R^(N1)is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, or optionally substituted aryl (e.g.,wherein R^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆ alkyl,such as methyl, ethyl, isopropyl, or n-propyl)); and each Y⁴ is,independently, H, hydroxy, thiol, optionally substituted alkyl,optionally substituted alkoxy, optionally substituted thioalkoxy,optionally substituted alkoxyalkoxy, or optionally substituted amino.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), thering including U is in the β-D (e.g., β-D-ribo) configuration.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), thering including U is in the α-L (e.g., α-L-ribo) configuration.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), one ormore B is not pseudouridine (ψ) or 5-methyl-cytidine (m⁵C). In someembodiments, about 10% to about 100% of n number of B nucleobases is notw or m⁵C (e.g., from 10% to 20%, from 10% to 35%, from 10% to 50%, from10% to 60%, from 10% to 75%, from 10% to 90%, from 10% to 95%, from 10%to 98%, from 10% to 99%, from 20% to 35%, from 20% to 50%, from 20% to60%, from 20% to 75%, from 20% to 90%, from 20% to 95%, from 20% to 98%,from 20% to 99%, from 20% to 100%, from 50% to 60%, from 50% to 75%,from 50% to 90%, from 50% to 95%, from 50% to 98%, from 50% to 99%, from50% to 100%, from 75% to 90%, from 75% to 95%, from 75% to 98%, from 75%to 99%, and from 75% to 100% of n number of B is not ψ or m⁵C). In someembodiments, B is not ψ or m⁵C.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), when Bis an unmodified nucleobase selected from cytosine, guanine, uracil andadenine, then at least one of Y′, Y², or Y³ is not O.

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a modified ribose. In some embodiments, the polynucleotide,primary construct, or mmRNA (e.g., the first region, the first flankingregion, or the second flanking region) includes n number of linkednucleosides having Formula (IIa)-(IIc):

or a pharmaceutically acceptable salt or stereoisomer thereof. Inparticular embodiments, U is O or C(R^(U))_(nu), wherein nu is aninteger from 0 to 2 and each R^(U) is, independently, H, halo, oroptionally substituted alkyl (e.g., U is —CH₂— or —CH—). In otherembodiments, each of R¹, R², R³, R⁴, and R⁵ is, independently, H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, or absent (e.g., each R¹ and R² is,independently, H, halo, hydroxy, optionally substituted alkyl, oroptionally substituted alkoxy; each R³ and R⁴ is, independently, H oroptionally substituted alkyl; and R⁵ is H or hydroxy), and

is a single bond or double bond.

In particular embodiments, the polynucleotidesor mmRNA includes n numberof linked nucleosides having Formula (IIb-1)-(IIb-2):

or a pharmaceutically acceptable salt or stereoisomer thereof. In someembodiments, U is O or C(R^(U))_(nu), wherein nu is an integer from 0 to2 and each R^(U) is, independently, H, halo, or optionally substitutedalkyl (e.g., U is —CH₂— or —CH—). In other embodiments, each of R¹ andR² is, independently, H, halo, hydroxy, thiol, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, or absent(e.g., each R¹ and R² is, independently, H, halo, hydroxy, optionallysubstituted alkyl, or optionally substituted alkoxy, e.g., H, halo,hydroxy, alkyl, or alkoxy). In particular embodiments, R² is hydroxy oroptionally substituted alkoxy (e.g., methoxy, ethoxy, or any describedherein).

In particular embodiments, the polynucleotide, primary construct, ormmRNA includes n number of linked nucleosides having Formula(IIc-1)-(IIc-4):

or a pharmaceutically acceptable salt or stereoisomer thereof. In someembodiments, U is O or C(R^(U))_(nu), wherein nu is an integer from 0 to2 and each R^(U) is, independently, H, halo, or optionally substitutedalkyl (e.g., U is —CH₂— or —CH—). In some embodiments, each of R¹, R²,and R³ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, or absent(e.g., each R¹ and R² is, independently, H, halo, hydroxy, optionallysubstituted alkyl, or optionally substituted alkoxy, e.g., H, halo,hydroxy, alkyl, or alkoxy; and each R³ is, independently, H oroptionally substituted alkyl)). In particular embodiments, R² isoptionally substituted alkoxy (e.g., methoxy or ethoxy, or any describedherein). In particular embodiments, R¹ is optionally substituted alkyl,and R² is hydroxy. In other embodiments, R¹ is hydroxy, and R² isoptionally substituted alkyl. In further embodiments, R³ is optionallysubstituted alkyl.

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes an acyclic modified ribose. In some embodiments, thepolynucleotide, primary construct, or mmRNA (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides having Formula (IId)-(IIf):

or a pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes an acyclic modified hexitol. In some embodiments, thepolynucleotide, primary construct, or mmRNA (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides Formula (IIg)-(IIj):

or a pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a sugar moiety having a contracted or an expanded ribose ring.In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, the first flanking region, or the secondflanking region) includes n number of linked nucleosides having Formula

(IIk)-(IIm):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of R^(1′), R^(1″), R^(2′), and R^(2″) is, independently, H, halo,hydroxy, optionally substituted alkyl, optionally substituted alkoxy,optionally substituted alkenyloxy, optionally substituted alkynyloxy,optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy,or absent; and wherein the combination of R^(2′) and R³ or thecombination of R^(2″) and R³ can be taken together to form optionallysubstituted alkylene or optionally substituted heteroalkylene.

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a locked modified ribose. In some embodiments, thepolynucleotide, primary construct, or mmRNA (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides having Formula (IIn):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(3′) is O, S, or —NR^(N1)—, wherein R^(N1) is H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl and R³″ isoptionally substituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)or optionally substituted heteroalkylene (e.g., —CH₂NH—, —CH₂CH₂NH—,—CH₂OCH₂—, or —CH₂CH₂OCH₂—)(e.g., R^(3′) is O and R^(3″) is optionallysubstituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)).

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes n number of linked nucleosides having Formula (IIn-1)-(II-n2):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(3′) is O, S, or —NR^(N1)—, wherein R^(N1) is H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl and R^(3″) isoptionally substituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)or optionally substituted heteroalkylene (e.g., —CH₂NH—, —CH₂CH₂NH—,—CH₂OCH₂—, or —CH₂CH₂OCH₂—) (e.g., R^(3′) is O and R^(3″) is optionallysubstituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)).

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a locked modified ribose that forms a tetracyclic heterocyclyl.In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, the first flanking region, or the secondflanking region) includes n number of linked nucleosides having Formula(IIo):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(12a), R^(12c), T^(1′), T^(1″), T^(2′), T^(2″), V¹, and V³ are asdescribed herein.

Any of the formulas for the polynucleotides, primary constructs, ormmRNA can include one or more nucleobases described herein (e.g.,Formulas (b1)-(b43)).

In one embodiment, the present invention provides methods of preparing apolynucleotide, primary construct, or mmRNA, wherein the polynucleotidecomprises n number of nucleosides having Formula (Ia), as definedherein:

the method comprising reacting a compound of Formula (Ma), as definedherein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a polynucleotide, primary construct, or mmRNA comprising atleast one nucleotide (e.g., mmRNA molecule), the method comprising:reacting a compound of Formula (Ma), as defined herein, with a primer, acDNA template, and an RNA polymerase.

In one embodiment, the present invention provides methods of preparing apolynucleotide, primary construct, or mmRNA comprising at least onenucleotide (e.g., mmRNA molecule), wherein the polynucleotide comprisesn number of nucleosides having Formula (Ia), as defined herein:

the method comprising reacting a compound of Formula (IIIa-1), asdefined herein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a polynucleotide, primary construct, or mmRNA comprising atleast one nucleotide (e.g., mmRNA molecule), the method comprising:

reacting a compound of Formula (IIIa-1), as defined herein, with aprimer, a cDNA template, and an RNA polymerase.

In one embodiment, the present invention provides methods of preparing amodified mRNA comprising at least one nucleotide (e.g., mmRNA molecule),wherein the polynucleotide comprises n number of nucleosides havingFormula (Ia-2), as defined herein:

the method comprising reacting a compound of Formula (IIIa-2), asdefined herein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a modified mRNA comprising at least one nucleotide (e.g.,mmRNA molecule), the method comprising:

reacting a compound of Formula (IIIa-2), as defined herein, with aprimer, a cDNA template, and an RNA polymerase.

In some embodiments, the reaction may be repeated from 1 to about 7,000times. In any of the embodiments herein, B may be a nucleobase ofFormula (b1)-(b43).

The polynucleotides, primary constructs, and mmRNA can optionallyinclude 5′ and/or 3′ flanking regions, which are described herein.

Modified RNA (mmRNA) Molecules

The present invention also includes building blocks, e.g., modifiedribonucleosides, modified ribonucleotides, of modified RNA (mmRNA)molecules. For example, these building blocks can be useful forpreparing the polynucleotides, primary constructs, or mmRNA of theinvention.

In some embodiments, the building block molecule has Formula (Ma) or(IIIa-1):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinthe substituents are as described herein (e.g., for Formula (Ia) and(Ia-1)), and wherein when B is an unmodified nucleobase selected fromcytosine, guanine, uracil and adenine, then at least one of Y¹, Y², orY³ is not O.

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IVa)-(IVb):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, Formula (IVa) or (IVb) is combined with a modified uracil(e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), suchas formula (b1), (b8), (b28), (b29), or (b30)). In particularembodiments, Formula (IVa) or (IVb) is combined with a modified cytosine(e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36),such as formula (b10) or (b32)). In particular embodiments, Formula(IVa) or (IVb) is combined with a modified guanine (e.g., any one offormulas (b15)-(b17) and (b37)-(b40)). In particular embodiments,Formula (IVa) or (IVb) is combined with a modified adenine (e.g., anyone of formulas (b18)-(b20) and (b41)-(b43)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IVc)-(IVk):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IVc)-(IVk) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IVc)-(IVk) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IVc)-(IVk) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IVc)-(IVk) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (Va) or (Vb):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IXa)-(IXd):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXa)-(IXd) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXa)-(IXd) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXa)-(IXd) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXa)-(IXd) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IXe)-(IXg):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXe)-(IXg) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula ( )), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXe)-(IXg) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXe)-(IXg) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXe)-(IXg) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IXh)-(IXk):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXh)-(IXk) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXh)-(IXk) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXh)-(IXk) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXh)-(IXk) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IX1)-(IXr):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r1 and r2 is, independently, an integer from 0 to 5 (e.g., from 0to 3, from 1 to 3, or from 1 to 5) and B is as described herein (e.g.,any one of (b1)-(b43)). In particular embodiments, one of Formulas(IX1)-(IXr) is combined with a modified uracil (e.g., any one offormulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1),(b8), (b28), (b29), or (b30)). In particular embodiments, one ofFormulas (IX1)-(IXr) is combined with a modified cytosine (e.g., any oneof formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula(b10) or (b32)). In particular embodiments, one of Formulas (IX1)-(IXr)is combined with a modified guanine (e.g., any one of formulas(b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas(IX1)-(IXr) is combined with a modified adenine (e.g., any one offormulas (b18)-(b20) and (b41)-(b43)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can beselected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can beselected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5) and s1 is as described herein.

In some embodiments, the building block molecule, which may beincorporated into a nucleic acid (e.g., RNA, mRNA, polynucleotide,primary construct, or mmRNA), is a modified uridine (e.g., selected fromthe group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, is amodified cytidine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)). For example, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, is amodified adenosine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, is amodified guanosine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the chemical modification can include replacementof C group at C-5 of the ring (e.g., for a pyrimidine nucleoside, suchas cytosine or uracil) with N (e.g., replacement of the >CH group at C-5with >NR^(N1) group, wherein R^(N)1 is H or optionally substitutedalkyl). For example, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In another embodiment, the chemical modification can include replacementof the hydrogen at C-5 of cytosine with halo (e.g., Br, Cl, F, or I) oroptionally substituted alkyl (e.g., methyl). For example, the buildingblock molecule, which may be incorporated into a polynucleotide, primaryconstruct, or mmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In yet a further embodiment, the chemical modification can include afused ring that is formed by the NH₂ at the C-4 position and the carbonatom at the C-5 position. For example, the building block molecule,which may be incorporated into a polynucleotide, primary construct, ormmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

Modifications on the Sugar

The modified nucleosides and nucleotides (e.g., building blockmolecules), which may be incorporated into a polynucleotide, primaryconstruct, or mmRNA (e.g., RNA or mRNA, as described herein), can bemodified on the sugar of the ribonucleic acid. For example, the 2′hydroxyl group (OH) can be modified or replaced with a number ofdifferent substituents. Exemplary substitutions at the 2′-positioninclude, but are not limited to, H, halo, optionally substituted C₁₋₆alkyl; optionally substituted C₁₋₆ alkoxy; optionally substituted C₆₋₁₀aryloxy; optionally substituted C₃₋₈ cycloalkyl; optionally substitutedC₃₋₈ cycloalkoxy; optionally substituted C₆₋₁₀ aryloxy; optionallysubstituted C₆₋₁₀ aryl-C₁₋₆ alkoxy, optionally substituted C₁₋₁₂(heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any describedherein); a polyethyleneglycol (PEG), —O(CH₂CH₂O)_(n)CH₂CH₂OR, where R isH or optionally substituted alkyl, and n is an integer from 0 to 20(e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4,from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from4 to 10, from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA)in which the 2′-hydroxyl is connected by a C₁₋₆ alkylene or C₁₋₆heteroalkylene bridge to the 4′-carbon of the same ribose sugar, whereexemplary bridges included methylene, propylene, ether, or aminobridges; aminoalkyl, as defined herein; aminoalkoxy, as defined herein;amino as defined herein; and amino acid, as defined herein

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting modified nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino that also has a phosphoramidate backbone); multicyclic forms(e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA)(e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attachedto phosphodiester bonds), threose nucleic acid (TNA, where ribose isreplace with α-L-threofuranosyl-(3′→2′)), and peptide nucleic acid (PNA,where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone). The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a polynucleotide, primaryconstruct, or mmRNA molecule can include nucleotides containing, e.g.,arabinose, as the sugar.

Modifications on the Nucleobase

The present disclosure provides for modified nucleosides andnucleotides. As described herein “nucleoside” is defined as a compoundcontaining a sugar molecule (e.g., a pentose or ribose) or a derivativethereof in combination with an organic base (e.g., a purine orpyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein, “nucleotide” is defined as anucleoside including a phosphate group. The modified nucleotides may bysynthesized by any useful method, as described herein (e.g., chemically,enzymatically, or recombinantly to include one or more modified ornon-natural nucleosides).

The modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil.

The modified nucleosides and nucleotides can include a modifiednucleobase. Examples of nucleobases found in RNA include, but are notlimited to, adenine, guanine, cytosine, and uracil. Examples ofnucleobase found in DNA include, but are not limited to, adenine,guanine, cytosine, and thymine. These nucleobases can be modified orwholly replaced to provide polynucleotides, primary constructs, or mmRNAmolecules having enhanced properties, e.g., resistance to nucleasesthrough disruption of the binding of a major groove binding partner.Table 8 below identifies the chemical faces of each canonicalnucleotide. Circles identify the atoms comprising the respectivechemical regions.

Watson-Crick Major Groove Minor Groove Base-pairing Face Face FacePyrimidines Cytidine:

Uridine:

Purines Adenosine:

Guanosine:

In some embodiments, B is a modified uracil. Exemplary modified uracilsinclude those having Formula (b1)-(b5):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

is a single or double bond;

each of T^(1′), T^(1″), T^(2′), and T^(2″) is, independently, H,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy, or the combination of T^(1′) andT^(1″) or the combination of T^(2′) and T^(2″) join together (e.g., asin T²) to form O (oxo), S (thio), or Se (seleno);

each of V¹ and V² is, independently, O, S, N(R^(Vb))_(nv), orC(R^(Vb))_(nv), wherein nv is an integer from 0 to 2 and each R^(Vb) is,independently, H, halo, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,optionally substituted aminoalkyl (e.g., substituted with anN-protecting group, such as any described herein, e.g.,trifluoroacetyl), optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g.,substituted with an N-protecting group, such as any described herein,e.g., trifluoroacetyl), optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, or optionally substituted alkynyloxy (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl);

R¹⁰ is H, halo, optionally substituted amino acid, hydroxy, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aminoalkyl, optionallysubstituted hydroxyalkyl, optionally substituted hydroxyalkenyl,optionally substituted hydroxyalkynyl, optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted alkoxy, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy,optionally substituted carboxyalkoxy, optionally substitutedcarboxyalkyl, or optionally substituted carbamoylalkyl;

R¹¹ is H or optionally substituted alkyl;

R^(12a) is H, optionally substituted alkyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, or optionally substitutedaminoalkynyl, optionally substituted carboxyalkyl (e.g., optionallysubstituted with hydroxy), optionally substituted carboxyalkoxy,optionally substituted carboxyaminoalkyl, or optionally substitutedcarbamoylalkyl; and

R^(12c) is H, halo, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted thioalkoxy, optionally substituted amino,optionally substituted hydroxyalkyl, optionally substitutedhydroxyalkenyl, optionally substituted hydroxyalkynyl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl.

Other exemplary modified uracils include those having Formula (b6)-(b9):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

is a single or double bond;

each of T^(1′), T^(1″), T^(2′), and T^(2″) is, independently, H,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy, or the combination of T^(1′) andT^(1″) join together (e.g., as in T¹) or the combination of T^(2′) andT^(2″) join together (e.g., as in T²) to form O (oxo), S (thio), or Se(seleno), or each T¹ and T² is, independently, O (oxo), S (thio), or Se(seleno);

each of W¹ and W² is, independently, N(R^(Wa))_(nw) or C(R^(Wa))_(nw),an integer from 0 to 2 and each R^(Wa) is, independently, H, optionallysubstituted alkyl, or optionally substituted alkoxy;

each V³ is, independently, O, S, N(R^(Va))_(nv), or C(R^(Va))_(nv),wherein nv is an integer from 0 to 2 and each R^(Va) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted hydroxyalkyl, optionally substitutedhydroxyalkenyl, optionally substituted hydroxyalkynyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclyl, optionally substituted alkheterocyclyl,optionally substituted alkoxy, optionally substituted alkenyloxy, oroptionally substituted alkynyloxy, optionally substituted aminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted acylaminoalkyl (e.g., substituted with an N-protectinggroup, such as any described herein, e.g., trifluoroacetyl), optionallysubstituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl), and wherein R^(Va) and R^(12c)taken together with the carbon atoms to which they are attached can formoptionally substituted cycloalkyl, optionally substituted aryl, oroptionally substituted heterocyclyl (e.g., a 5- or 6-membered ring);

R^(12a) is H, optionally substituted alkyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, optionally substituted carboxyalkyl (e.g., optionallysubstituted with hydroxy and/or an O-protecting group), optionallysubstituted carboxyalkoxy, optionally substituted carboxyaminoalkyl,optionally substituted carbamoylalkyl, or absent;

R^(12b) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, optionally substituted alkaryl, optionally substitutedheterocyclyl, optionally substituted alkheterocyclyl, optionallysubstituted amino acid, optionally substituted alkoxycarbonylacyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedalkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl,optionally substituted alkoxycarbonylalkynyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl,

wherein the combination of R^(12b) and T^(1′) or the combination ofR^(12b) and R^(12c) can join together to form optionally substitutedheterocyclyl; and

R^(12c) is H, halo, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted thioalkoxy, optionally substituted amino,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,or optionally substituted aminoalkynyl.

Further exemplary modified uracils include those having Formula(b28)-(b31):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of T¹ and T² is, independently, O (oxo), S (thio), or Se (seleno);

each R^(Vb′) and R^(Vb″) is, independently, H, halo, optionallysubstituted amino acid, optionally substituted alkyl, optionallysubstituted haloalkyl, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted acylaminoalkyl (e.g., substituted with an N-protectinggroup, such as any described herein, e.g., trifluoroacetyl), optionallysubstituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl) (e.g., R^(vb′) is optionallysubstituted alkyl, optionally substituted alkenyl, or optionallysubstituted aminoalkyl, e.g., substituted with an N-protecting group,such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl);

R^(12a) is H, optionally substituted alkyl, optionally substitutedcarboxyaminoalkyl, optionally substituted aminoalkyl (e.g., e.g.,substituted with an N-protecting group, such as any described herein,e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, or optionally substituted aminoalkynyl; and

R^(12b) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substituted aminoalkynyl(e.g., e.g., substituted with an N-protecting group, such as anydescribed herein, e.g., trifluoroacetyl, or sulfoalkyl),

optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy,optionally substituted carboxyalkoxy, optionally substitutedcarboxyalkyl, or optionally substituted carbamoylalkyl.

In particular embodiments, T¹ is O (oxo), and T² is S (thio) or Se(seleno). In other embodiments, T¹ is S (thio), and T² is O (oxo) or Se(seleno). In some embodiments, R^(Vb′) is H, optionally substitutedalkyl, or optionally substituted alkoxy.

In other embodiments, each R^(12a) and R^(12b) is, independently, H,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted hydroxyalkyl. Inparticular embodiments, R^(12a) is H. In other embodiments, both R^(12a)and R^(12b) are H.

In some embodiments, each R^(Vb′) of R^(12b) is, independently,optionally substituted aminoalkyl (e.g., substituted with anN-protecting group, such as any described herein, e.g., trifluoroacetyl,or sulfoalkyl), optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, or optionally substituted acylaminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl). In some embodiments, the amino and/oralkyl of the optionally substituted aminoalkyl is substituted with oneor more of optionally substituted alkyl, optionally substituted alkenyl,optionally substituted sulfoalkyl, optionally substituted carboxy (e.g.,substituted with an O-protecting group), optionally substituted hydroxy(e.g., substituted with an O-protecting group), optionally substitutedcarboxyalkyl (e.g., substituted with an O-protecting group), optionallysubstituted alkoxycarbonylalkyl (e.g., substituted with an O-protectinggroup), or N-protecting group. In some embodiments, optionallysubstituted aminoalkyl is substituted with an optionally substitutedsulfoalkyl or optionally substituted alkenyl. In particular embodiments,R^(12a) and R^(Vb″) are both H. In particular embodiments, T¹ is O(oxo), and T² is S (thio) or Se (seleno).

In some embodiments, R^(Vb′) is optionally substitutedalkoxycarbonylalkyl or optionally substituted carbamoylalkyl.

In particular embodiments, the optional substituent for R^(12a),R^(12b), R^(12c), or R^(Va) is a polyethylene glycol group (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl); or an amino-polyethylene glycol group (e.g.,—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl).

In some embodiments, B is a modified cytosine. Exemplary modifiedcytosines include compounds of Formula (b10)-(b14):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of T^(3′) and T^(3″) is, independently, H, optionally substitutedalkyl, optionally substituted alkoxy, or optionally substitutedthioalkoxy, or the combination of T^(3′) and T^(3″) join together (e.g.,as in T³) to form O (oxo), S (thio), or Se (seleno);

each V⁴ is, independently, O, S, N(R^(Vc))_(nv), or C(R^(Vc))_(nv),wherein nv is an integer from 0 to 2 and each R^(Vc) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionallysubstituted with any substituent described herein, such as thoseselected from (1)-(21) for alkyl), wherein the combination of R^(13b)and R^(Vc) can be taken together to form optionally substitutedheterocyclyl;

each V⁵ is, independently, N(R^(Vd))_(nv), or C(R^(Vd))_(nv), wherein nvis an integer from 0 to 2 and each R^(Vd) is, independently, H, halo,optionally substituted amino acid, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionallysubstituted with any substituent described herein, such as thoseselected from (1)-(21) for alkyl) (e.g., V⁵ is —CH or N);

each of R^(13a) and R^(13b) is, independently, H, optionally substitutedacyl, optionally substituted acyloxyalkyl, optionally substituted alkyl,or optionally substituted alkoxy, wherein the combination of R^(13b) andR¹⁴ can be taken together to form optionally substituted heterocyclyl;

each R¹⁴ is, independently, H, halo, hydroxy, thiol, optionallysubstituted acyl, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted hydroxyalkyl (e.g., substituted with an O-protecting group),optionally substituted hydroxyalkenyl, optionally substitutedhydroxyalkynyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedacyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H,alkyl, aryl, or phosphoryl), azido, optionally substituted aryl,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkyl; and

each of R¹⁵ and R¹⁶ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl.

Further exemplary modified cytosines include those having Formula(b32)-(b35):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of T¹ and T³ is, independently, O (oxo), S (thio), or Se (seleno);

each of R^(13a) and R^(13b) is, independently, H, optionally substitutedacyl, optionally substituted acyloxyalkyl, optionally substituted alkyl,or optionally substituted alkoxy, wherein the combination of R^(13b) andR¹⁴ can be taken together to form optionally substituted heterocyclyl;

each R¹⁴ is, independently, H, halo, hydroxy, thiol, optionallysubstituted acyl, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted hydroxyalkyl (e.g., substituted with an O-protecting group),optionally substituted hydroxyalkenyl, optionally substitutedhydroxyalkynyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedacyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H,alkyl, aryl, or phosphoryl), azido, optionally substituted aryl,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, optionally substituted aminoalkyl (e.g., hydroxyalkyl,alkyl, alkenyl, or alkynyl), optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl; and

each of R¹⁵ and R¹⁶ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl (e.g.,R¹⁵ is H, and R¹⁶ is H or optionally substituted alkyl).

In some embodiments, R¹⁵ is H, and R¹⁶ is H or optionally substitutedalkyl. In particular embodiments, R¹⁴ is H, acyl, or hydroxyalkyl. Insome embodiments, R¹⁴ is halo. In some embodiments, both R¹⁴ and R¹⁵ areH. In some embodiments, both R¹⁵ and R¹⁶ are H. In some embodiments,each of R¹⁴ and R¹⁵ and R¹⁶ is H. In further embodiments, each ofR^(13a) and R^(13b) is independently, H or optionally substituted alkyl.

Further non-limiting examples of modified cytosines include compounds ofFormula (b36):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each R^(13b) is, independently, H, optionally substituted acyl,optionally substituted acyloxyalkyl, optionally substituted alkyl, oroptionally substituted alkoxy, wherein the combination of R^(13b) andR^(14b) can be taken together to form optionally substitutedheterocyclyl;

each R^(14a) and R^(14b) is, independently, H, halo, hydroxy, thiol,optionally substituted acyl, optionally substituted amino acid,optionally substituted alkyl, optionally substituted haloalkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted hydroxyalkyl (e.g., substituted with anO-protecting group), optionally substituted hydroxyalkenyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted aminoalkoxy, optionallysubstituted alkoxyalkoxy, optionally substituted acyloxyalkyl,optionally substituted amino (e.g., —NHR, wherein R is H, alkyl, aryl,phosphoryl, optionally substituted aminoalkyl, or optionally substitutedcarboxyaminoalkyl), azido, optionally substituted aryl, optionallysubstituted heterocyclyl, optionally substituted alkheterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,or optionally substituted aminoalkynyl; and

each of R¹⁵ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl.

In particular embodiments, R^(14b) is an optionally substituted aminoacid (e.g., optionally substituted lysine). In some embodiments, R^(14a)is H.

In some embodiments, B is a modified guanine Exemplary modified guaninesinclude compounds of Formula (b15)-(b17):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of T^(4′), T^(4″), T^(5′), T^(5″), T^(6′), and T^(6″) isindependently, H, optionally substituted alkyl, or optionallysubstituted alkoxy, and wherein the combination of T^(4′) and T^(4″)(e.g., as in T⁴) or the combination of T^(5′) and T^(5″) (e.g., as inT⁵) or the combination of T^(6′) and T^(6″) (e.g., as in T⁶) jointogether form O (oxo), S (thio), or Se (seleno);

each of V⁵ and V⁶ is, independently, O, S, N(R^(Vd))_(nv), orC(R^(Vd))_(nv), wherein nv is an integer from 0 to 2 and each R^(Vd) is,independently, H, halo, thiol, optionally substituted amino acid, cyano,amidine, optionally substituted aminoalkyl, optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, or optionally substituted alkynyloxy (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl), optionally substitutedthioalkoxy, or optionally substituted amino; and

each of R¹⁷, R¹⁸, R^(19a), R^(19b), R²¹, R²², R²³, and R²⁴ is,independently, H, halo, thiol, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted thioalkoxy, optionally substituted amino, or optionallysubstituted amino acid.

Exemplary modified guanosines include compounds of Formula (b37)-(b40):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of T^(4′) is, independently, H, optionally substituted alkyl, oroptionally substituted alkoxy, and each T⁴ is, independently, O (oxo), S(thio), or Se (seleno);

each of R¹⁸, R^(19a), R^(19b), and R²¹ is, independently, H, halo,thiol, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted thioalkoxy,optionally substituted amino, or optionally substituted amino acid.

In some embodiments, R¹⁸ is H or optionally substituted alkyl. Infurther embodiments, T⁴ is oxo. In some embodiments, each of R^(19a) andR^(19b) is, independently, H or optionally substituted alkyl.

In some embodiments, B is a modified adenine. Exemplary modifiedadenines include compounds of Formula (b18)-(b20):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each V⁷ is, independently, O, S, N(R^(Ve))_(nv), or C(R^(Ve))_(nv),wherein nv is an integer from 0 to 2 and each R^(Ve) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy, oroptionally substituted alkynyloxy (e.g., optionally substituted with anysubstituent described herein, such as those selected from (1)-(21) foralkyl);

each R²⁵ is, independently, H, halo, thiol, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted thioalkoxy, or optionally substituted amino;

each of R^(26a) and R^(26b) is, independently, H, optionally substitutedacyl, optionally substituted amino acid, optionally substitutedcarbamoylalkyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted alkoxy, orpolyethylene glycol group (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl); or an amino-polyethylene glycol group(e.g., —NR^(Ni)(CH₂)_(s2)(CH₂CH₂O)_(si)(CH₂)_(s3)NR^(N1), wherein s1 isan integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1)is, independently, hydrogen or optionally substituted C₁₋₆ alkyl);

each R²⁷ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted thioalkoxy or optionallysubstituted amino;

each R²⁸ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, or optionally substituted alkynyl; and

each R²⁹ is, independently, H, optionally substituted acyl, optionallysubstituted amino acid, optionally substituted carbamoylalkyl,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted alkoxy, or optionallysubstituted amino.

Exemplary modified adenines include compounds of Formula (b41)-(b43):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each R²⁵ is, independently, H, halo, thiol, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted thioalkoxy, or optionally substituted amino;

each of R^(26a) and R^(26b) is, independently, H, optionally substitutedacyl, optionally substituted amino acid, optionally substitutedcarbamoylalkyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted alkoxy, orpolyethylene glycol group (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(si)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl); or an amino-polyethylene glycol group(e.g., —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 isan integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1)is, independently, hydrogen or optionally substituted C₁₋₆ alkyl); and

each R²⁷ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted thioalkoxy, or optionallysubstituted amino.

In some embodiments, R^(26a) is H, and R^(26b) is optionally substitutedalkyl. In some embodiments, each of R^(26a) and R^(26b) is,independently, optionally substituted alkyl. In particular embodiments,R²⁷ is optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy. In other embodiments, R²⁵ isoptionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy.

In particular embodiments, the optional substituent for R^(26a),R^(26b), or R²⁹ is a polyethylene glycol group (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl); or an amino-polyethylene glycol group (e.g.,—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl).

In some embodiments, B may have Formula (b21):

wherein X¹² is, independently, O, S, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene, xa is aninteger from 0 to 3, and R^(12a) and T² are as described herein.

In some embodiments, B may have Formula (b22):

wherein R^(10′) is, independently, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, optionally substituted alkoxy,optionally substituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedcarboxyalkoxy, optionally substituted carboxyalkyl, or optionallysubstituted carbamoylalkyl, and R¹¹, R^(12a), T¹, and T² are asdescribed herein.

In some embodiments, B may have Formula (b23):

wherein R¹⁰ is optionally substituted heterocyclyl (e.g., optionallysubstituted furyl, optionally substitued thienyl, or optionallysubstitued pyrrolyl), optionally substituted aryl (e.g., optionallysubstituted phenyl or optionally substituted naphthyl), or anysubstituent described herein (e.g., for R¹⁰); and wherein R¹¹ (e.g., Hor any substituent described herein), R^(12a) (e.g., H or anysubstituent described herein), T¹ (e.g., oxo or any substituentdescribed herein), and T² (e.g., oxo or any substituent describedherein) are as described herein.In some embodiments, B may have Formula (b24):

wherein R^(14′) is, independently, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted alkaryl, optionally substituted alkheterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, optionally substituted alkoxy,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedcarboxyalkoxy, optionally substituted carboxyalkyl, or optionallysubstituted carbamoylalkyl, and R^(13a), R^(13b), R¹⁵ and T³ are asdescribed herein.

In some embodiments, B may have Formula (b25):

wherein R^(14′) is optionally substituted heterocyclyl (e.g., optionallysubstituted furyl, optionally substitued thienyl, or optionallysubstitued pyrrolyl), optionally substituted aryl (e.g., optionallysubstituted phenyl or optionally substituted naphthyl), or anysubstituent described herein (e.g., for R¹⁴ or R^(14′)); and whereinR^(13a) (e.g., H or any substituent described herein), R^(13b) (e.g., Hor any substituent described herein), R¹⁵ (e.g., H or any substituentdescribed herein), and T³ (e.g., oxo or any substituent describedherein) are as described herein.

In some embodiments, B is a nucleobase selected from the groupconsisting of cytosine, guanine, adenine, and uracil. In someembodiments, B may be:

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm⁵s²U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e.,having the nucleobase deoxythymine), 1-methylpseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine(m¹s⁴ψ)^(,) 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine (also known as1-methylpseudouridine (m¹ψ)), 3-β-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵ Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm), N4,2′-O-dimethyl-cytidine (m⁴ Cm), 5-formyl-2′-O-methyl-cytidine(f⁵ Cm), N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂ Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N-6-isopentenyl-adenosine (ms²¹⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m′Am), 2′-β-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanineExemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o₂yW), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (O),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m^(2,7)G),N2, N2,7-dimethyl-guanosine (m^(2,2,7)G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m′Im), and 2′-β-ribosylguanosine(phosphate) (Gr(p)).

The nucleobase of the nucleotide can be independently selected from apurine, a pyrimidine, a purine or pyrimidine analog. For example, thenucleobase can each be independently selected from adenine, cytosine,guanine, uracil, or hypoxanthine. In another embodiment, the nucleobasecan also include, for example, naturally-occurring and syntheticderivatives of a base, including pyrazolo[3,4-d]pyrimidines,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine,deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides aredepicted using the shorthand A, G, C, T or U, each letter refers to therepresentative base and/or derivatives thereof, e.g., A includes adenineor adenine analogs, e.g., 7-deaza adenine).

Modifications on the Internucleoside Linkage

The modified nucleotides, which may be incorporated into apolynucleotide, primary construct, or mmRNA molecule, can be modified onthe internucleoside linkage (e.g., phosphate backbone). Herein, in thecontext of the polynucleotide backbone, the phrases “phosphate” and“phosphodiester” are used interchangeably. Backbone phosphate groups canbe modified by replacing one or more of the oxygen atoms with adifferent substituent. Further, the modified nucleosides and nucleotidescan include the wholesale replacement of an unmodified phosphate moietywith another internucleoside linkage as described herein. Examples ofmodified phosphate groups include, but are not limited to,phosphorothioate, phosphoroselenates, boranophosphates,boranophosphateesters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates,alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioateshave both non-linking oxygens replaced by sulfur. The phosphate linkercan also be modified by the replacement of a linking oxygen withnitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates),and carbon (bridged methylene-phosphonates).

The α-thio substituted phosphate moiety is provided to confer stabilityto RNA and DNA polymers through the unnatural phosphorothioate backbonelinkages. Phosphorothioate DNA and RNA have increased nucleaseresistance and subsequently a longer half-life in a cellularenvironment. Phosphorothioate linked polynucleotides, primaryconstructs, or mmRNA molecules are expected to also reduce the innateimmune response through weaker binding/activation of cellular innateimmune molecules.

In specific embodiments, a modified nucleoside includes analpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine,5′-O-(1-thiophosphate)-cytidine (α-thio-cytidine),5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or5′-O-(1-thiophosphate)-pseudouridine).

Other internucleoside linkages that may be employed according to thepresent invention, including internucleoside linkages which do notcontain a phosphorous atom, are described herein below.

Combinations of Modified Sugars, Nucleobases, and InternucleosideLinkages

The polynucleotides, primary constructs, and mmRNA of the invention caninclude a combination of modifications to the sugar, the nucleobase,and/or the internucleoside linkage. These combinations can include anyone or more modifications described herein. For examples, any of thenucleotides described herein in Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr) can be combined with any of the nucleobasesdescribed herein (e.g., in Formulas (b1)-(b43) or any other describedherein).

Synthesis of Polypeptides, Primary Constructs, And mmRNA Molecules

The polypeptides, primary constructs, and mmRNA molecules for use inaccordance with the invention may be prepared according to any usefultechnique, as described herein. The modified nucleosides and nucleotidesused in the synthesis of polynucleotides, primary constructs, and mmRNAmolecules disclosed herein can be prepared from readily availablestarting materials using the following general methods and procedures.Where typical or preferred process conditions (e.g., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are provided, a skilled artisan would be able to optimize anddevelop additional process conditions. Optimum reaction conditions mayvary with the particular reactants or solvent used, but such conditionscan be determined by one skilled in the art by routine optimizationprocedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry(e.g., UV-visible), or mass spectrometry, or by chromatography such ashigh performance liquid chromatography (HPLC) or thin layerchromatography.

Preparation of polypeptides, primary constructs, and mmRNA molecules ofthe present invention can involve the protection and deprotection ofvarious chemical groups. The need for protection and deprotection, andthe selection of appropriate protecting groups can be readily determinedby one skilled in the art. The chemistry of protecting groups can befound, for example, in Greene, et al., Protective Groups in OrganicSynthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein byreference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents, which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Resolution of racemic mixtures of modified nucleosides and nucleotidescan be carried out by any of numerous methods known in the art. Anexample method includes fractional recrystallization using a “chiralresolving acid” which is an optically active, salt-forming organic acid.Suitable resolving agents for fractional recrystallization methods are,for example, optically active acids, such as the D and L forms oftartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelicacid, malic acid, lactic acid or the various optically activecamphorsulfonic acids. Resolution of racemic mixtures can also becarried out by elution on a column packed with an optically activeresolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elutionsolvent composition can be determined by one skilled in the art.

Modified nucleosides and nucleotides (e.g., building block molecules)can be prepared according to the synthetic methods described in Ogata etal., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res.22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568(1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each ofwhich are incorporated by reference in their entirety.

The polypeptides, primary constructs, and mmRNA of the invention may ormay not be uniformly modified along the entire length of the molecule.For example, one or more or all types of nucleotide (e.g., purine orpyrimidine, or any one or more or all of A, G, U, C) may or may not beuniformly modified in a polynucleotide of the invention, or in a givenpredetermined sequence region thereof (e.g. one or more of the sequenceregions represented in FIG. 1). In some embodiments, all nucleotides Xin a polynucleotide of the invention (or in a given sequence regionthereof) are modified, wherein X may any one of nucleotides A, G, U, C,or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U,A+G+C, G+U+C or A+G+C.

Different sugar modifications, nucleotide modifications, and/orinternucleoside linkages (e.g., backbone structures) may exist atvarious positions in the polynucleotide, primary construct, or mmRNA.One of ordinary skill in the art will appreciate that the nucleotideanalogs or other modification(s) may be located at any position(s) of apolynucleotide, primary construct, or mmRNA such that the function ofthe polynucleotide, primary construct, or mmRNA is not substantiallydecreased. A modification may also be a 5′ or 3′ terminal modification.The polynucleotide, primary construct, or mmRNA may contain from about1% to about 100% modified nucleotides (either in relation to overallnucleotide content, or in relation to one or more types of nucleotide,i.e. any one or more of A, G, U or C) or any intervening percentage(e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%,from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10%to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100%).

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a modified pyrimidine (e.g., a modified uracil/uridine/U ormodified cytosine/cytidine/C). In some embodiments, the uracil oruridine (generally: U) in the polynucleotide, primary construct, ormmRNA molecule may be replaced with from about 1% to about 100% of amodified uracil or modified uridine (e.g., from 1% to 20%, from 1% to25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100% of a modified uracil or modifieduridine). The modified uracil or uridine can be replaced by a compoundhaving a single unique structure or by a plurality of compounds havingdifferent structures (e.g., 2, 3, 4 or more unique structures, asdescribed herein). In some embodiments, the cytosine or cytidine(generally: C) in the polynucleotide, primary construct, or mmRNAmolecule may be replaced with from about 1% to about 100% of a modifiedcytosine or modified cytidine (e.g., from 1% to 20%, from 1% to 25%,from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1%to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%,from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%,from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50%to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to100%, and from 95% to 100% of a modified cytosine or modified cytidine).The modified cytosine or cytidine can be replaced by a compound having asingle unique structure or by a plurality of compounds having differentstructures (e.g., 2, 3, 4 or more unique structures, as describedherein).

In some embodiments, the present disclosure provides methods ofsynthesizing a polynucleotide, primary construct, or mmRNA (e.g., thefirst region, first flanking region, or second flanking region)including n number of linked nucleosides having Formula (Ia-1):

comprising:a) reacting a nucleotide of Formula (IV-1):

with a phosphoramidite compound of Formula (V-1):

wherein Y⁹ is H, hydroxy, phosphoryl, pyrophosphate, sulfate, amino,thiol, optionally substituted amino acid, or a peptide (e.g., includingfrom 2 to 12 amino acids); and each P¹, P², and P³ is, independently, asuitable protecting group; and

denotes a solid support;to provide a polynucleotide, primary construct, or mmRNA of Formula(VI-1):

andb) oxidizing or sulfurizing the polynucleotide, primary construct, ormmRNA of Formula (V) to yield a polynucleotide, primary construct, ormmRNA of Formula (VII-1):

andc) removing the protecting groups to yield the polynucleotide, primaryconstruct, or mmRNA of Formula (Ia).

In some embodiments, steps a) and b) are repeated from 1 to about 10,000times. In some embodiments, the methods further comprise a nucleotide(e.g., mmRNA molecule) selected from the group consisting of A, C, G andU adenosine, cytosine, guanosine, and uracil. In some embodiments, thenucleobase may be a pyrimidine or derivative thereof. In someembodiments, the polynucleotide, primary construct, or mmRNA istranslatable.

Other components of polynucleotides, primary constructs, and mmRNA areoptional, and are beneficial in some embodiments. For example, a 5′untranslated region (UTR) and/or a 3′UTR are provided, wherein either orboth may independently contain one or more different nucleotidemodifications. In such embodiments, nucleotide modifications may also bepresent in the translatable region. Also provided are polynucleotides,primary constructs, and mmRNA containing a Kozak sequence.

Exemplary syntheses of modified nucleotides, which are incorporated intoa modified nucleic acid or mmRNA, e.g., RNA or mRNA, are provided belowin Scheme 1 through Scheme 11. Scheme 1 provides a general method forphosphorylation of nucleosides, including modified nucleosides.

Various protecting groups may be used to control the reaction. Forexample, Scheme 2 provides the use of multiple protecting anddeprotecting steps to promote phosphorylation at the 5′ position of thesugar, rather than the 2′ and 3′ hydroxyl groups.

Modified nucleotides can be synthesized in any useful manner. Schemes 3,4, and 7 provide exemplary methods for synthesizing modified nucleotideshaving a modified purine nucleobase; and Schemes 5 and 6 provideexemplary methods for synthesizing modified nucleotides having amodified pseudouridine or pseudoisocytidine, respectively.

Schemes 8 and 9 provide exemplary syntheses of modified nucleotides.Scheme 10 provides a non-limiting biocatalytic method for producingnucleotides.

Scheme 11 provides an exemplary synthesis of a modified uracil, wherethe N1 position is modified with R^(12b), as provided elsewhere, and the5′-position of ribose is phosphorylated. T¹, T², R^(12a), R^(12b), and rare as provided herein. This synthesis, as well as optimized versionsthereof, can be used to modify other pyrimidine nucleobases and purinenucleobases (see e.g., Formulas (b1)-(b43)) and/or to install one ormore phosphate groups (e.g., at the 5′ position of the sugar). Thisalkylating reaction can also be used to include one or more optionallysubstituted alkyl group at any reactive group (e.g., amino group) in anynucleobase described herein (e.g., the amino groups in the Watson-Crickbase-pairing face for cytosine, uracil, adenine, and guanine)

Combinations of Nucleotides in mmRNA

Further examples of modified nucleotides and modified nucleotidecombinations are provided below in Table 9. These combinations ofmodified nucleotides can be used to form the polypeptides, primaryconstructs, or mmRNA of the invention. Unless otherwise noted, themodified nucleotides may be completely substituted for the naturalnucleotides of the modified nucleic acids or mmRNA of the invention. Asa non-limiting example, the natural nucleotide uridine may besubstituted with a modified nucleoside described herein. In anothernon-limiting example, the natural nucleotide uridine may be partiallysubstituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%)with at least one of the modified nucleoside disclosed herein.

TABLE 9 Modified Nucleotide Modified Nucleotide Combinationα-thio-cytidine α-thio-cytidine/5-iodo-uridineα-thio-cytidine/N1-methyl-pseudouridine α-thio-cytidine/α-thio-uridineα-thio-cytidine/5-methyl-uridine α-thio-cytidine/pseudo-uridine about50% of the cytosines are α-thio-cytidine pseudoiso-pseudoisocytidine/5-iodo-uridine cytidinepseudoisocytidine/N1-methyl-pseudouridinepseudoisocytidine/α-thio-uridine pseudoisocytidine/5-methyl-uridinepseudoisocytidine/pseudouridine about 25% of cytosines arepseudoisocytidine pseudoisocytidine/about 50% of uridines are N1-methyl-pseudouridine and about 50% of uridines are pseudo- uridinepseudoisocytidine/about 25% of uridines are N1-methyl- pseudouridine andabout 25% of uridines are pseudo- uridine pyrrolo-pyrrolo-cytidine/5-iodo-uridine cytidinepyrrolo-cytidine/N1-methyl-pseudouridine pyrrolo-cytidine/α-thio-uridinepyrrolo-cytidine/5-methyl-uridine pyrrolo-cytidine/pseudouridine about50% of the cytosines are pyrrolo-cytidine 5-methyl-5-methyl-cytidine/5-iodo-uridine cytidine5-methyl-cytidine/N1-methyl-pseudouridine5-methyl-cytidine/α-thio-uridine 5-methyl-cytidine/5-methyl-uridine5-methyl-cytidine/pseudouridine about 25% of cytosines are5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridinesare 2-thio- uridine about 50% of uridines are 5-methyl-cytidine/about50% of uridines are 2-thio-uridine N4-acetyl-N4-acetyl-cytidine/5-iodo-uridine cytidineN4-acetyl-cytidine/N1-methyl-pseudouridineN4-acetyl-cytidine/α-thio-uridine N4-acetyl-cytidine/5-methyl-uridineN4-acetyl-cytidine/pseudouridine about 50% of cytosines areN4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidineN4-acetyl-cytidine/5-methoxy-uridine N4-acetyl-cytidine/5-bromo-uridineN4-acetyl-cytidine/2-thio-uridine about 50% of cytosines areN4-acetyl-cytidine/about 50% of uridines are 2-thio-uridine

Further examples of modified nucleotide combinations are provided belowin Table 10. These combinations of modified nucleotides can be used toform the polypeptides, primary constructs, or mmRNA of the invention.

TABLE 10 Modified Nucleotide Modified Nucleotide Combination modifiedcytidine modified cytidine with (b10)/pseudouridine having one ormodified cytidine with (b10)/N1-methyl-pseudouridine more nucleobasesmodified cytidine with (b10)/5-methoxy-uridine of Formula (b10) modifiedcytidine with (b10)/5-methyl-uridine modified cytidine with(b10)/5-bromo-uridine modified cytidine with (b10)/2-thio-uridine about50% of cytidine substituted with modified cytidine (b10)/about 50% ofuridines are 2-thio- uridine modified cytidine modified cytidine with(b32)/pseudouridine having one or modified cytidine with(b32)/N1-methyl-pseudouridine more nucleobases modified cytidine with(b32)/5-methoxy-uridine of Formula (b32) modified cytidine with(b32)/5-methyl-uridine modified cytidine with (b32)/5-bromo-uridinemodified cytidine with (b32)/2-thio-uridine about 50% of cytidinesubstituted with modified cytidine (b32)/about 50% of uridines are2-thio- uridine modified uridine modified uridine with(b1)/N4-acetyl-cytidine having one or modified uridine with(b1)/5-methyl-cytidine more nucleobases of Formula (b1) modified uridinemodified uridine with (b8)/N4-acetyl-cytidine having one or modifieduridine with (b8)/5-methyl-cytidine more nucleobases of Formula (b8)modified uridine modified uridine with (b28)/N4-acetyl-cytidine havingone or modified uridine with (b28)/5-methyl-cytidine more nucleobases ofFormula (b28) modified uridine modified uridine with(b29)/N4-acetyl-cytidine having one or modified uridine with(b29)/5-methyl-cytidine more nucleobases of Formula (b29) modifieduridine modified uridine with (b30)/N4-acetyl-cytidine having one ormodified uridine with (b30)/5-methyl-cytidine more nucleobases ofFormula (b30)

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14) (e.g., at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the uracils are replaced by acompound of Formula (b1)-(b9) (e.g., at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14), and at least 25% of the uracils arereplaced by a compound of Formula (b1)-(b9) (e.g., at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%).

IV. Pharmaceutical Compositions Formulation, Administration, Deliveryand Dosing

The present invention provides polynucleotides, primary constructs andmmRNA compositions and complexes in combination with one or morepharmaceutically acceptable excipients. Pharmaceutical compositions mayoptionally comprise one or more additional active substances, e.g.therapeutically and/or prophylactically active substances. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents may be found, for example, in Remington: The Science and Practiceof Pharmacy 21^(st) ed., Lippincott Williams & Wilkins, 2005(incorporated herein by reference).

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to polynucleotides, primaryconstructs and mmRNA to be delivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

Formulations

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using one or more excipients to: (1) increase stability; (2)increase cell transfection; (3) permit the sustained or delayed release(e.g., from a depot formulation of the polynucleotide, primaryconstruct, or mmRNA); (4) alter the biodistribution (e.g., target thepolynucleotide, primary construct, or mmRNA to specific tissues or celltypes); (5) increase the translation of encoded protein in vivo; and/or(6) alter the release profile of encoded protein in vivo. In addition totraditional excipients such as any and all solvents, dispersion media,diluents, or other liquid vehicles, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, excipients of the present invention can include,without limitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with polynucleotide, primary construct, or mmRNA (e.g., fortransplantation into a subject), hyaluronidase, nanoparticle mimics andcombinations thereof. Accordingly, the formulations of the invention caninclude one or more excipients, each in an amount that togetherincreases the stability of the polynucleotide, primary construct, ormmRNA, increases cell transfection by the polynucleotide, primaryconstruct, or mmRNA, increases the expression of polynucleotide, primaryconstruct, or mmRNA encoded protein, and/or alters the release profileof polynucleotide, primary construct, or mmRNA encoded proteins.Further, the primary construct and mmRNA of the present invention may beformulated using self-assembled nucleic acid nanoparticles.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient may generally be equal to the dosage of theactive ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage including, but not limited to,one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition maycomprise between 0.1% and 99% (w/w) of the active ingredient.

In some embodiments, the formulations described herein may contain atleast one mmRNA. As a non-limiting example, the formulations may contain1, 2, 3, 4 or 5 mmRNA. In one embodiment the formulation may containmodified mRNA encoding proteins selected from categories such as, butnot limited to, human proteins, veterinary proteins, bacterial proteins,biological proteins, antibodies, immunogenic proteins, therapeuticpeptides and proteins, secreted proteins, plasma membrane proteins,cytoplasmic and cytoskeletal proteins, intrancellular membrane boundproteins, nuclear proteins, proteins associated with human diseaseand/or proteins associated with non-human diseases. In one embodiment,the formulation contains at least three modified mRNA encoding proteins.In one embodiment, the formulation contains at least five modified mRNAencoding proteins.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but is notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, andthe like, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro,Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference in its entirety). The use of a conventionalexcipient medium may be contemplated within the scope of the presentdisclosure, except insofar as any conventional excipient medium may beincompatible with a substance or its derivatives, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticalcomposition.

In some embodiments, the particle size of the lipid nanoparticle may beincreased and/or decreased. The change in particle size may be able tohelp counter biological reaction such as, but not limited to,inflammation or may increase the biological effect of the modified mRNAdelivered to mammals.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, surface active agents and/or emulsifiers, preservatives,buffering agents, lubricating agents, and/or oils. Such excipients mayoptionally be included in the pharmaceutical formulations of theinvention.

Lipidoids

The synthesis of lipidoids has been extensively described andformulations containing these compounds are particularly suited fordelivery of polynucleotides, primary constructs or mmRNA (see Mahon etal., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med.2010 267:9-21; Akinc et al., Nat. Biotechnol. 2008 26:561-569; Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., ProcNatl Acad Sci USA. 2011 108:12996-3001; all of which are incorporatedherein in their entireties).

While these lipidoids have been used to effectively deliver doublestranded small interfering RNA molecules in rodents and non-humanprimates (see Akinc et al., Nat. Biotechnol. 2008 26:561-569;Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920;Akinc et al., Mol. Ther. 2009 17:872-879; Love et al., Proc Natl AcadSci USA. 2010 107:1864-1869; Leuschner et al., Nat. Biotechnol. 201129:1005-1010; all of which is incorporated herein in their entirety),the present disclosure describes their formulation and use in deliveringsingle stranded polynucleotides, primary constructs, or mmRNA.Complexes, micelles, liposomes or particles can be prepared containingthese lipidoids and therefore, can result in an effective delivery ofthe polynucleotide, primary construct, or mmRNA, as judged by theproduction of an encoded protein, following the injection of a lipidoidformulation via localized and/or systemic routes of administration.Lipidoid complexes of polynucleotides, primary constructs, or mmRNA canbe administered by various means including, but not limited to,intravenous, intramuscular, or subcutaneous routes.

In vivo delivery of nucleic acids may be affected by many parameters,including, but not limited to, the formulation composition, nature ofparticle PEGylation, degree of loading, oligonucleotide to lipid ratio,and biophysical parameters such as, but not limited to, particle size(Akinc et al., Mol. Ther. 2009 17:872-879; herein incorporated byreference in its entirety). As an example, small changes in the anchorchain length of poly(ethylene glycol) (PEG) lipids may result insignificant effects on in vivo efficacy. Formulations with the differentlipidoids, including, but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry,401:61 (2010); herein incorporated by reference in its entirety),C12-200 (including derivatives and variants), and MD1, can be tested forin vivo activity.

The lipidoid referred to herein as “98N12-5” is disclosed by Akinc etal., Mol. Ther. 2009 17:872-879 and is incorporated by reference in itsentirety. (See FIG. 2)

The lipidoid referred to herein as “C12-200” is disclosed by Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 (see FIG. 2) and Liu andHuang, Molecular Therapy. 2010 669-670 (see FIG. 2); both of which areherein incorporated by reference in their entirety. The lipidoidformulations can include particles comprising either 3 or 4 or morecomponents in addition to polynucleotide, primary construct, or mmRNA.As an example, formulations with certain lipidoids, include, but are notlimited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and10% PEG (C₁₋₄ alkyl chain length). As another example, formulations withcertain lipidoids, include, but are not limited to, C12-200 and maycontain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5%cholesterol, and 1.5% PEG-DMG.

In one embodiment, a polynucleotide, primary construct, or mmRNAformulated with a lipidoid for systemic intravenous administration cantarget the liver. For example, a final optimized intravenous formulationusing polynucleotide, primary construct, or mmRNA, and comprising alipid molar composition of 42% 98N12-5, 48% cholesterol, and 10%PEG-lipid with a final weight ratio of about 7.5 to 1 total lipid topolynucleotide, primary construct, or mmRNA, and a C₁₋₄ alkyl chainlength on the PEG lipid, with a mean particle size of roughly 50-60 nm,can result in the distribution of the formulation to be greater than 90%to the liver.(see, Akinc et al., Mol. Ther. 2009 17:872-879; hereinincorporated by reference in its entirety). In another example, anintravenous formulation using a C12-200 (see U.S. provisionalapplication 61/175,770 and published international applicationWO2010129709, each of which is herein incorporated by reference in theirentirety) lipidoid may have a molar ratio of 50/10/38.5/1.5 ofC12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG, with a weightratio of 7 to 1 total lipid to polynucleotide, primary construct, ormmRNA, and a mean particle size of 80 nm may be effective to deliverpolynucleotide, primary construct, or mmRNA to hepatocytes (see, Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 herein incorporated byreference in its entirety). In another embodiment, an MD1lipidoid-containing formulation may be used to effectively deliverpolynucleotide, primary construct, or mmRNA to hepatocytes in vivo. Thecharacteristics of optimized lipidoid formulations for intramuscular orsubcutaneous routes may vary significantly depending on the target celltype and the ability of formulations to diffuse through theextracellular matrix into the blood stream. While a particle size ofless than 150 nm may be desired for effective hepatocyte delivery due tothe size of the endothelial fenestrae (see, Akinc et al., Mol. Ther.2009 17:872-879 herein incorporated by reference in its entirety), useof a lipidoid-formulated polynucleotide, primary construct, or mmRNA todeliver the formulation to other cells types including, but not limitedto, endothelial cells, myeloid cells, and muscle cells may not besimilarly size-limited. Use of lipidoid formulations to deliver siRNA invivo to other non-hepatocyte cells such as myeloid cells and endotheliumhas been reported (see Akinc et al., Nat. Biotechnol. 2008 26:561-569;Leuschner et al., Nat. Biotechnol. 2011 29:1005-1010; Cho et al. Adv.Funct. Mater. 2009 19:3112-3118; 8^(th) International Judah FolkmanConference, Cambridge, Mass. Oct. 8-9, 2010; each of which is hereinincorporated by reference in its entirety). Effective delivery tomyeloid cells, such as monocytes, lipidoid formulations may have asimilar component molar ratio. Different ratios of lipidoids and othercomponents including, but not limited to, disteroylphosphatidyl choline,cholesterol and PEG-DMG, may be used to optimize the formulation of thepolynucleotide, primary construct, or mmRNA for delivery to differentcell types including, but not limited to, hepatocytes, myeloid cells,muscle cells, etc. For example, the component molar ratio may include,but is not limited to, 50% C12-200, 10% disteroylphosphatidyl choline,38.5% cholesterol, and %1.5 PEG-DMG (see Leuschner et al., NatBiotechno12011 29:1005-1010; herein incorporated by reference in itsentirety). The use of lipidoid formulations for the localized deliveryof nucleic acids to cells (such as, but not limited to, adipose cellsand muscle cells) via either subcutaneous or intramuscular delivery, maynot require all of the formulation components desired for systemicdelivery, and as such may comprise only the lipidoid and thepolynucleotide, primary construct, or mmRNA.

Combinations of different lipidoids may be used to improve the efficacyof polynucleotide, primary construct, or mmRNA directed proteinproduction as the lipidoids may be able to increase cell transfection bythe polynucleotide, primary construct, or mmRNA; and/or increase thetranslation of encoded protein (see Whitehead et al., Mol. Ther. 2011,19:1688-1694, herein incorporated by reference in its entirety).

Liposomes, Lipoplexes, and Lipid Nanoparticles

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using one or more liposomes, lipoplexes, or lipidnanoparticles. In one embodiment, pharmaceutical compositions ofpolynucleotide, primary construct, or mmRNA include liposomes. Liposomesare artificially-prepared vesicles which may primarily be composed of alipid bilayer and may be used as a delivery vehicle for theadministration of nutrients and pharmaceutical formulations. Liposomescan be of different sizes such as, but not limited to, a multilamellarvesicle (MLV) which may be hundreds of nanometers in diameter and maycontain a series of concentric bilayers separated by narrow aqueouscompartments, a small unicellular vesicle (SUV) which may be smallerthan 50 nm in diameter, and a large unilamellar vesicle (LUV) which maybe between 50 and 500 nm in diameter. Liposome design may include, butis not limited to, opsonins or ligands in order to improve theattachment of liposomes to unhealthy tissue or to activate events suchas, but not limited to, endocytosis. Liposomes may contain a low or ahigh pH in order to improve the delivery of the pharmaceuticalformulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes which may deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat. Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;all of which are incorporated herein in their entireties). The originalmanufacture method by Wheeler et al. was a detergent dialysis method,which was later improved by Jeffs et al. and is referred to as thespontaneous vesicle formation method. The liposome formulations arecomposed of 3 to 4 lipid components in addition to the polynucleotide,primary construct, or mmRNA. As an example a liposome can contain, butis not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline(DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane(DODMA), as described by Jeffs et al. As another example, certainliposome formulations may contain, but are not limited to, 48%cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where thecationic lipid can be 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA),DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA),as described by Heyes et al.

In one embodiment, pharmaceutical compositions may include liposomeswhich may be formed to deliver mmRNA which may encode at least oneimmunogen. The mmRNA may be encapsulated by the liposome and/or it maybe contained in an aqueous core which may then be encapsulated by theliposome (see International Pub. Nos. WO2012031046, WO2012031043,WO2012030901 and WO2012006378; each of which is herein incorporated byreference in their entirety). In another embodiment, the mmRNA which mayencode an immunogen may be formulated in a cationic oil-in-wateremulsion where the emulsion particle comprises an oil core and acationic lipid which can interact with the mmRNA anchoring the moleculeto the emulsion particle (see International Pub. No. WO2012006380;herein incorporated by reference in its entirety). In yet anotherembodiment, the lipid formulation may include at least cationic lipid, alipid which may enhance transfection and a least one lipid whichcontains a hydrophilic head group linked to a lipid moiety(International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582; eachof which is herein incorporated by reference in their entirety). Inanother embodiment, the polynucleotides, primary constructs and/or mmRNAencoding an immunogen may be formulated in a lipid vesicle which mayhave crosslinks between functionalized lipid bilayers (see U.S. Pub. No.20120177724, herein incorporated by reference in its entirety).

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be formulated in a lipid vesicle which may have crosslinks betweenfunctionalized lipid bilayers.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be formulated in a liposome comprising a cationic lipid. Theliposome may have a molar ratio of nitrogen atoms in the cationic lipidto the phophates in the RNA (N:P ratio) of between 1:1 and 20:1 asdescribed in International Publication No. WO2013006825, hereinincorporated by reference in its entirety. In another embodiment, theliposome may have a N:P ratio of greater than 20:1 or less than 1:1.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be formulated in a lipid-polycation complex. The formation of thelipid-polycation complex may be accomplished by methods known in the artand/or as described in U.S. Pub. No. 20120178702, herein incorporated byreference in its entirety. As a non-limiting example, the polycation mayinclude a cationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine and the cationic peptidesdescribed in International Pub. No. WO2012013326; herein incorporated byreference in its entirety. In another embodiment, the polynucleotides,primary constructs and/or mmRNA may be formulated in a lipid-polycationcomplex which may further include a neutral lipid such as, but notlimited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

The liposome formulation may be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176; herein incorporated byreference in its entirety), the liposome formulation was composed of57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3%cholesterol, and 1.4% PEG-c-DMA. As another example, changing thecomposition of the cationic lipid could more effectively deliver siRNAto various antigen presenting cells (Basha et al. Mol. Ther. 201119:2186-2200; herein incorporated by reference in its entirety).

In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP)formulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the LNP formulations. As anon-limiting example, LNP formulations may contain 1-5% of the lipidmolar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC andcholesterol. In another embodiment the PEG-c-DOMG may be replaced with aPEG lipid such as, but not limited to, PEG- DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In one embodiment, the polynucleotides, primary constructs or mmRNA maybe formulated in a lipid nanoparticle such as those described inInternational Publication No. WO2012170930, herein incorporated byreference in its entirety.

In one embodiment, the cationic lipid may be selected from, but notlimited to, a cationic lipid described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724,WO201021865 and WO2008103276, U.S. Pat. Nos. 7,893,302, 7,404,969 and8,283,333 and US Patent Publication No. US20100036115 and US20120202871;each of which is herein incorporated by reference in their entirety. Inanother embodiment, the cationic lipid may be selected from, but notlimited to, formula A described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365 and WO2012044638; each of whichis herein incorporated by reference in their entirety. In yet anotherembodiment, the cationic lipid may be selected from, but not limited to,formula CLI-CLXXIX of International Publication No. WO2008103276,formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formula CLI-CLXXXXII ofU.S. Pat. No. 7,404,969 and formula I-V1 of US Patent Publication No.US20100036115; each of which is herein incorporated by reference intheir entirety. As a non-limiting example, the cationic lipid may beselected from (20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine,(1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine,(18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine,(18Z)-N,N-dimethylheptacos-18-en-10-amine,(17Z)-N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,(20Z)-N,N-dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)-N,N-dimethylnonacos-17-en-10-amine,(24Z)-N,N-dimethyltritriacont-24-en-10-amine,(20Z)-N,N-dimethylnonacos-20-en-10-amine,(22Z)-N,N-dimethylhentriacont-22-en-10-amine,(16Z)-N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl] eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-o ctylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcycIopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecyIcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl} dodecan-1-amine,1-[(1R,2S)-2-hepty lcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amineand (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine or apharmaceutically acceptable salt or stereoisomer thereof.

In one embodiment, the lipid may be a cleavable lipid such as thosedescribed in International Publication No. WO2012170889, hereinincorporated by reference in its entirety.

In one embodiment, the cationic lipid may be synthesized by methodsknown in the art and/or as described in International Publication Nos.WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913,WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 andWO201021865; each of which is herein incorporated by reference in theirentirety.

In one embodiment, the LNP formulations of the polynucleotides, primaryconstructs and/or mmRNA may contain PEG-c-DOMG at 3% lipid molar ratio.In another embodiment, the LNP formulations polynucleotides, primaryconstructs and/or mmRNA may contain PEG-c-DOMG at 1.5% lipid molarratio.

In one embodiment, the pharmaceutical compositions of thepolynucleotides, primary constructs and/or mmRNA may include at leastone of the PEGylated lipids described in International Publication No.2012099755, herein incorporated by reference.

In one embodiment, the LNP formulation may contain PEG-DMG 2000(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethyleneglycol)-2000).

In one embodiment, the LNP formulation may contain PEG-DMG 2000, acationic lipid known in the art and at least one other component. Inanother embodiment, the LNP formulation may contain PEG-DMG 2000, acationic lipid known in the art, DSPC and cholesterol. As a non-limitingexample, the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPCand cholesterol. As another non-limiting example the LNP formulation maycontain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of2:40:10:48 (see e.g., Geall et al., Nonviral delivery of self-amplifyingRNA vaccines, PNAS 2012; PMID: 22908294; herein incorporated byreference in its entirety). As another non-limiting example, modifiedRNA described herein may be formulated in a nanoparticle to be deliveredby a parenteral route as described in U.S. Pub. No. 20120207845; hereinincorporated by reference in its entirety.

In one embodiment, the LNP formulation may be formulated by the methodsdescribed in International Publication Nos. WO2011127255 orWO2008103276, each of which is herein incorporated by reference in theirentirety. As a non-limiting example, modified RNA described herein maybe encapsulated in LNP formulations as described in WO2011127255 and/orWO2008103276; each of which is herein incorporated by reference in theirentirety.

In one embodiment, LNP formulations described herein may comprise apolycationic composition. As a non-limiting example, the polycationiccomposition may be selected from formula 1-60 of US Patent PublicationNo. US20050222064; herein incorporated by reference in its entirety. Inanother embodiment, the LNP formulations comprising a polycationiccomposition may be used for the delivery of the modified RNA describedherein in vivo and/or in vitro.

In one embodiment, the LNP formulations described herein mayadditionally comprise a permeability enhancer molecule. Non-limitingpermeability enhancer molecules are described in US Patent PublicationNo. US20050222064; herein incorporated by reference in its entirety.

In one embodiment, the pharmaceutical compositions may be formulated inliposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech,Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutralDOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,siRNA delivery for ovarian cancer (Landen et al. Cancer Biology &Therapy 2006 5(12)1708-1713); herein incorporated by reference in itsentirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

The nanoparticle formulations may be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a modified nucleic acid molecule(e.g., mmRNA). As a non-limiting example, the carbohydrate carrier mayinclude, but is not limited to, an anhydride-modified phytoglycogen orglycogen-type material, phtoglycogen octenyl succinate, phytoglycogenbeta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g.,International Publication No. WO2012109121; herein incorporated byreference in its entirety).

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In one embodiment, the internal ester linkage may be located on eitherside of the saturated carbon. Non-limiting examples of reLNPs include,

In one embodiment, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805; each of which is herein incorporated byreference in their entirety). The polymer may encapsulate thenanospecies or partially encapsulate the nanospecies. The immunogen maybe a recombinant protein, a modified RNA and/or a primary constructdescribed herein. In one embodiment, the lipid nanoparticle may beformulated for use in a vaccine such as, but not limited to, against apathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosla tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; each of which is herein incorporated by reference in theirentirety). The transport of nanoparticles may be determined using ratesof permeation and/or fluorescent microscopy techniques including, butnot limited to, fluorescence recovery after photobleaching (FRAP) andhigh resolution multiple particle tracking (MPT). As a non-limitingexample, compositions which can penetrate a mucosal barrier may be madeas described in U.S. Pat. No. 8,241,670, herein incorporated byreference in its entirety.

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. The polymeric material mayadditionally be irradiated. As a non-limiting example, the polymericmaterial may be gamma irradiated (See e.g., International App. No.WO201282165, herein incorporated by reference in its entirety).Non-limiting examples of specific polymers include poly(caprolactone)(PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA),poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lacticacid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid)(PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA),poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer (suchas a branched polyether-polyamide block copolymer described inInternational Publication No. WO2013012476, herein incorporated byreference in its entirety), and (poly(ethylene glycol))-(poly(propyleneoxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., USPublication 20120121718 and US Publication 20100003337 and U.S. Pat. No.8,263,665; each of which is herein incorporated by reference in theirentirety). The co-polymer may be a polymer that is generally regarded assafe (GRAS) and the formation of the lipid nanoparticle may be in such away that no new chemical entities are created. For example, the lipidnanoparticle may comprise poloxamers coating PLGA nanoparticles withoutforming new chemical entities which are still able to rapidly penetratehuman mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; hereinincorporated by reference in its entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, mmRNA, anionic proteins(e.g., bovine serum albumin), surfactants (e.g., cationic surfactantssuch as for example dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g.,N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol,sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosinβ4 dornase alfa, neltenexine, erdosteine) and various DNases includingrhDNase. The surface altering agent may be embedded or enmeshed in theparticle's surface or disposed (e.g., by coating, adsorption, covalentlinkage, or other process) on the surface of the lipid nanoparticle.(see e.g., US Publication 20100215580 and US Publication 20080166414;each of which is herein incorporated by reference in their entirety).

The mucus penetrating lipid nanoparticles may comprise at least onemmRNA described herein. The mmRNA may be encapsulated in the lipidnanoparticle and/or disposed on the surface of the paricle. The mmRNAmay be covalently coupled to the lipid nanoparticle. Formulations ofmucus penetrating lipid nanoparticles may comprise a plurality ofnanoparticles. Further, the formulations may contain particles which mayinteract with the mucus and alter the structural and/or adhesiveproperties of the surrounding mucus to decrease mucoadhesion which mayincrease the delivery of the mucus penetrating lipid nanoparticles tothe mucosal tissue.

In one embodiment, the polynucleotide, primary construct, or mmRNA isformulated as a lipoplex, such as, without limitation, the ATUPLEX™system, the DACC system, the DBTC system and other siRNA-lipoplextechnology from Silence Therapeutics (London, United Kingdom), STEMFECT™from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) orprotamine-based targeted and non-targeted delivery of nucleic acidsacids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al. IntJ Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 200613:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier etal., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al. MicrovascRes 2010 80:286-293Weide et al. J Immunother. 2009 32:498-507; Weide etal. J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther.4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15; Song etal., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl AcadSci USA. 2007 6; 104:4095-4100; deFougerolles Hum Gene Ther. 200819:125-132; all of which are incorporated herein by reference in itsentirety).

In one embodiment such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol. Ther. 201018:1357-1364; Song et al., Nat. Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety). One exampleof passive targeting of formulations to liver cells includes theDLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticleformulations which have been shown to bind to apolipoprotein E andpromote binding and uptake of these formulations into hepatocytes invivo (Akinc et al. Mol. Ther. 2010 18:1357-1364; herein incorporated byreference in its entirety). Formulations can also be selectivelytargeted through expression of different ligands on their surface asexemplified by, but not limited by, folate, transferrin,N-acetylgalactosamine (GalNAc), and antibody targeted approaches(Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchioand Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol MembrBiol. 2010 27:286-298; Patil et al., Crit. Rev Ther Drug Carrier Syst.2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhaoet al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol. Ther.2010 18:1357-1364; Srinivasan et al., Methods Mol. Biol. 2012820:105-116; Ben-Arie et al., Methods Mol. Biol. 2012 757:497-507; Peer2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA.2007 104:4095-4100; Kim et al., Methods Mol. Biol. 2011 721:339-353;Subramanya et al., Mol. Ther. 2010 18:2028-2037; Song et al., Nat.Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630;Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all of which areincorporated herein by reference in its entirety).

In one embodiment, the polynucleotide, primary construct, or mmRNA isformulated as a solid lipid nanoparticle. A solid lipid nanoparticle(SLN) may be spherical with an average diameter between 10 to 1000 nm.SLN possess a solid lipid core matrix that can solubilize lipophilicmolecules and may be stabilized with surfactants and/or emulsifiers. Ina further embodiment, the lipid nanoparticle may be a self-assemblylipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp1696-1702; herein incorporated by reference in its entirety).

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of polynucleotide, primary construct, or mmRNA directed proteinproduction as these formulations may be able to increase celltransfection by the polynucleotide, primary construct, or mmRNA; and/orincrease the translation of encoded protein. One such example involvesthe use of lipid encapsulation to enable the effective systemic deliveryof polyplex plasmid DNA (Heyes et al., Mol. Ther. 2007 15:713-720;herein incorporated by reference in its entirety). The liposomes,lipoplexes, or lipid nanoparticles may also be used to increase thestability of the polynucleotide, primary construct, or mmRNA.

In one embodiment, the polynucleotides, primary constructs, and/or themmRNA of the present invention can be formulated for controlled releaseand/or targeted delivery. As used herein, “controlled release” refers toa pharmaceutical composition or compound release profile that conformsto a particular pattern of release to effect a therapeutic outcome. Inone embodiment, the polynucleotides, primary constructs or the mmRNA maybe encapsulated into a delivery agent described herein and/or known inthe art for controlled release and/or targeted delivery. As used herein,the term “encapsulate” means to enclose, surround or encase. As itrelates to the formulation of the compounds of the invention,encapsulation may be substantial, complete or partial. The term“substitantially encapsulated” means that at least greater than 50, 60,70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999%of the pharmaceutical composition or compound of the invention may beenclosed, surrounded or encased within the delivery agent. “Partiallyencapsulation” means that less than 10, 10, 20, 30, 40 50 or less of thepharmaceutical composition or compound of the invention may be enclosed,surrounded or encased within the delivery agent. Advantageously,encapsulation may be determined by measuring the escape or the activityof the pharmaceutical composition or compound of the invention usingfluorescence and/or electron micrograph. For example, at least 1, 5, 10,20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 orgreater than 99.99% of the pharmaceutical composition or compound of theinvention are encapsulated in the delivery agent.

In one embodiment, the controlled release formulation may include, butis not limited to, tri-block co-polymers. As a non-limiting example, theformulation may include two different types of tri-block co-polymers(International Pub. No. WO2012131104 and WO2012131106; each of which isherein incorporated by reference in its entirety).

In another embodiment, the polynucleotides, primary constructs, or themmRNA may be encapsulated into a lipid nanoparticle or a rapidlyeliminated lipid nanoparticle and the lipid nanoparticles or a rapidlyeliminated lipid nanoparticle may then be encapsulated into a polymer,hydrogel and/or surgical sealant described herein and/or known in theart. As a non-limiting example, the polymer, hydrogel or surgicalsealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer,GELSITE®(Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (HalozymeTherapeutics, San Diego Calif.), surgical sealants such as fibrinogenpolymers (Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International,Inc Deerfield, Ill.), PEG-based sealants, and COSEAL® (BaxterInternational, Inc Deerfield, Ill.).

In another embodiment, the lipid nanoparticle may be encapsulated intoany polymer known in the art which may form a gel when injected into asubject. As another non-limiting example, the lipid nanoparticle may beencapsulated into a polymer matrix which may be biodegradable.

In one embodiment, the polynucleotide, primary construct, or mmRNAformulation for controlled release and/or targeted delivery may alsoinclude at least one controlled release coating. Controlled releasecoatings include, but are not limited to, OPADRY®,polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone,hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethylcellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such asethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).

In one embodiment, the controlled release and/or targeted deliveryformulation may comprise at least one degradable polyester which maycontain polycationic side chains. Degradeable polyesters include, butare not limited to, poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), and combinations thereof. In anotherembodiment, the degradable polyesters may include a PEG conjugation toform a PEGylated polymer.

In one embodiment, the polynucleotides, primary constructs, and/or themmRNA of the present invention may be encapsulated in a therapeuticnanoparticle. Therapeutic nanoparticles may be formulated by methodsdescribed herein and known in the art such as, but not limited to,International Pub Nos. WO2010005740, WO2010030763, WO2010005721,WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645,US20100087337, US20100068285, US20110274759, US20100068286 andUS20120288541 and U.S. Pat. Nos. 8,206,747, 8,293,276, 8,318,208 and8,318,211 each of which is herein incorporated by reference in theirentirety. In another embodiment, therapeutic polymer nanoparticles maybe identified by the methods described in US Pub No. US20120140790,herein incorporated by reference in its entirety.

In one embodiment, the therapeutic nanoparticle may be formulated forsustained release. As used herein, “sustained release” refers to apharmaceutical composition or compound that conforms to a release rateover a specific period of time. The period of time may include, but isnot limited to, hours, days, weeks, months and years. As a non-limitingexample, the sustained release nanoparticle may comprise a polymer and atherapeutic agent such as, but not limited to, the polynucleotides,primary constructs, and mmRNA of the present invention (seeInternational Pub No. 2010075072 and US Pub No. US20100216804,US20110217377 and US20120201859, each of which is herein incorporated byreference in their entirety).

In one embodiment, the therapeutic nanoparticles may be formulated to betarget specific. As a non-limiting example, the thereapeuticnanoparticles may include a corticosteroid (see International Pub. No.WO2011084518; herein incorporated by reference in its entirety). In oneembodiment, the therapeutic nanoparticles may be formulated to be cancerspecific. As a non-limiting example, the therapeutic nanoparticles maybe formulated in nanoparticles described in International Pub No.WO2008121949, WO2010005726, WO2010005725, WO2011084521 and US Pub No.US20100069426, US20120004293 and US20100104655, each of which is hereinincorporated by reference in their entirety.

In one embodiment, the nanoparticles of the present invention maycomprise a polymeric matrix. As a non-limiting example, the nanoparticlemay comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In one embodiment, the therapeutic nanoparticle comprises a diblockcopolymer. In one embodiment, the diblock copolymer may include PEG incombination with a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof.

As a non-limiting example the therapeutic nanoparticle comprises aPLGA-PEG block copolymer (see US Pub. No. US20120004293 and U.S. Pat.No. 8,236,330, each of which is herein incorporated by reference intheir entirety). In another non-limiting example, the therapeuticnanoparticle is a stealth nanoparticle comprising a diblock copolymer ofPEG and PLA or PEG and PLGA (see US Pat No 8,246,968 and InternationalPublication No. WO2012166923, each of which is herein incorporated byreference in its entirety).

In one embodiment, the therapeutic nanoparticle may comprise amultiblock copolymer (See e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910;each of which is herein incorporated by reference in its entirety).

In one embodiment, the block copolymers described herein may be includedin a polyion complex comprising a non-polymeric micelle and the blockcopolymer. (See e.g., U.S. Pub. No. 20120076836; herein incorporated byreference in its entirety).

In one embodiment, the therapeutic nanoparticle may comprise at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In one embodiment, the therapeutic nanoparticles may comprise at leastone cationic polymer described herein and/or known in the art.

In one embodiment, the therapeutic nanoparticles may comprise at leastone amine-containing polymer such as, but not limited to polylysine,polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters)(See e.g., U.S. Pat. No. 8,287,849; herein incorporated by reference inits entirety) and combinations thereof.

In one embodiment, the therapeutic nanoparticles may comprise at leastone degradable polyester which may contain polycationic side chains.Degradeable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In another embodiment, the degradablepolyesters may include a PEG conjugation to form a PEGylated polymer.

In another embodiment, the therapeutic nanoparticle may include aconjugation of at least one targeting ligand. The targeting ligand maybe any ligand known in the art such as, but not limited to, a monoclonalantibody. (Kirpotin et al, Cancer Res. 2006 66:6732-6740; hereinincorporated by reference in its entirety).

In one embodiment, the therapeutic nanoparticle may be formulated in anaqueous solution which may be used to target cancer (see InternationalPub No. WO2011084513 and US Pub No. US20110294717, each of which isherein incorporated by reference in their entirety).

In one embodiment, the polynucleotides, primary constructs, or mmRNA maybe encapsulated in, linked to and/or associated with syntheticnanocarriers. Synthetic nanocarriers include, but are not limited to,those described in International Pub. Nos. WO2010005740, WO2010030763,WO201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265,WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405,WO2012149411, WO2012149454 and WO2013019669, and US Pub. Nos.US20110262491, US20100104645, US20100087337 and US20120244222, each ofwhich is herein incorporated by reference in their entirety. Thesynthetic nanocarriers may be formulated using methods known in the artand/or described herein. As a non-limiting example, the syntheticnanocarriers may be formulated by the methods described in InternationalPub Nos. WO2010005740, WO2010030763 and WO201213501 and US Pub. Nos.US20110262491, US20100104645, US20100087337 and US2012024422, each ofwhich is herein incorporated by reference in their entirety. In anotherembodiment, the synthetic nanocarrier formulations may be lyophilized bymethods described in International Pub. No. WO2011072218 and U.S. Pat.No. 8,211,473; each of which is herein incorporated by reference intheir entirety.

In one embodiment, the synthetic nanocarriers may contain reactivegroups to release the polynucleotides, primary constructs and/or mmRNAdescribed herein (see International Pub. No. WO20120952552 and US PubNo. US20120171229, each of which is herein incorporated by reference intheir entirety).

In one embodiment, the synthetic nanocarriers may contain animmunostimulatory agent to enhance the immune response from delivery ofthe synthetic nanocarrier. As a non-limiting example, the syntheticnanocarrier may comprise a Th1 immunostimulatory agent which may enhancea Th1-based response of the immune system (see International Pub No.WO2010123569 and US Pub. No. US20110223201, each of which is hereinincorporated by reference in its entirety).

In one embodiment, the synthetic nanocarriers may be formulated fortargeted release. In one embodiment, the synthetic nanocarrier isformulated to release the polynucleotides, primary constructs and/ormmRNA at a specified pH and/or after a desired time interval. As anon-limiting example, the synthetic nanoparticle may be formulated torelease the polynucleotides, primary constructs and/or mmRNA after 24hours and/or at a pH of 4.5 (see International Pub. Nos. WO2010138193and WO2010138194 and US Pub Nos. US20110020388 and US20110027217, eachof which is herein incorporated by reference in their entireties).

In one embodiment, the synthetic nanocarriers may be formulated forcontrolled and/or sustained release of the polynucleotides, primaryconstructs and/or mmRNA described herein. As a non-limiting example, thesynthetic nanocarriers for sustained release may be formulated bymethods known in the art, described herein and/or as described inInternational Pub No. WO2010138192 and US Pub No. 20100303850, each ofwhich is herein incorporated by reference in their entirety.

In one embodiment, the synthetic nanocarrier may be formulated for useas a vaccine. In one embodiment, the synthetic nanocarrier mayencapsulate at least one polynucleotide, primary construct and/or mmRNAwhich encode at least one antigen. As a non-limiting example, thesynthetic nanocarrier may include at least one antigen and an excipientfor a vaccine dosage form (see International Pub No. WO2011150264 and USPub No. US20110293723, each of which is herein incorporated by referencein their entirety). As another non-limiting example, a vaccine dosageform may include at least two synthetic nanocarriers with the same ordifferent antigens and an excipient (see International Pub No.WO2011150249 and US Pub No. US20110293701, each of which is hereinincorporated by reference in their entirety). The vaccine dosage formmay be selected by methods described herein, known in the art and/ordescribed in International Pub No. WO2011150258 and US Pub No.US20120027806, each of which is herein incorporated by reference intheir entirety).

In one embodiment, the synthetic nanocarrier may comprise at least onepolynucleotide, primary construct and/or mmRNA which encodes at leastone adjuvant. As non-limiting example, the adjuvant may comprisedimethyldioctadecylammonium-bromide,dimethyldioctadecylammonium-chloride,dimethyldioctadecylammonium-phosphate ordimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or partof said apolar fraction of a total lipid extract of a mycobacterium (Seee.g, U.S. Pat. No. 8,241,610; herein incorporated by reference in itsentirety). In another embodiment, the synthetic nanocarrier may compriseat least one polynucleotide, primary construct and/or mmRNA and anadjuvant. As a non-limiting example, the synthetic nanocarriercomprising and adjuvant may be formulated by the methods described inInternational Pub No. WO2011150240 and US Pub No. US20110293700, each ofwhich is herein incorporated by reference in its entirety.

In one embodiment, the synthetic nanocarrier may encapsulate at leastone polynucleotide, primary construct and/or mmRNA which encodes apeptide, fragment or region from a virus. As a non-limiting example, thesynthetic nanocarrier may include, but is not limited to, thenanocarriers described in International Pub No. WO2012024621,WO201202629, WO2012024632 and US Pub No. US20120064110, US20120058153and US20120058154, each of which is herein incorporated by reference intheir entirety.

In one embodiment, the synthetic nanocarrier may be coupled to apolynucleotide, primary construct or mmRNA which may be able to triggera humoral and/or cytotoxic T lymphocyte (CTL) response (See e.g.,International Publication No. WO2013019669, herein incorporated byreference in its entirety).

In one embodiment, the nanoparticle may be optimized for oraladministration. The nanoparticle may comprise at least one cationicbiopolymer such as, but not limited to, chitosan or a derivativethereof. As a non-limiting example, the nanoparticle may be formulatedby the methods described in U.S. Pub. No. 20120282343; hereinincorporated by reference in its entirety. Polymers, BiodegradableNanoparticles, and Core-Shell Nanoparticles

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using natural and/or synthetic polymers. Non-limitingexamples of polymers which may be used for delivery include, but are notlimited to, DYNAMIC POLYCONJUGATE® (Arrowhead Reasearch Corp., Pasadena,Calif.) formulations from MIRUS® Bio (Madison, Wis.) and Roche Madison(Madison, Wis.), PHASERX™ polymer formulations such as, withoutlimitation, SMARTT POLYMER TECHNOLOGY™ (PHASERX®, Seattle, Wash.),DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego,Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena,Calif.), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers.RONDEL™ (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (ArrowheadResearch Corporation, Pasadena, Calif.) and pH responsive co-blockpolymers such as, but not limited to, PHASERX® (Seattle, Wash.).

A non-limiting example of chitosan formulation includes a core ofpositively charged chitosan and an outer portion of negatively chargedsubstrate (U.S. Pub. No. 20120258176; herein incorporated by referencein its entirety). Chitosan includes, but is not limited to N-trimethylchitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan(NPCS), EDTA-chitosan, low molecular weight chitosan, chitosanderivatives, or combinations thereof.

In one embodiment, the polymers used in the present invention haveundergone processing to reduce and/or inhibit the attachment of unwantedsubstances such as, but not limited to, bacteria, to the surface of thepolymer. The polymer may be processed by methods known and/or describedin the art and/or described in International Pub. No. WO2012150467,herein incorporated by reference in its entirety.

A non-limiting example of PLGA formulations include, but are not limitedto, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolvingPLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueoussolvent and leuprolide. Once injected, the PLGA and leuprolide peptideprecipitates into the subcutaneous space).

Many of these polymer approaches have demonstrated efficacy indelivering oligonucleotides in vivo into the cell cytoplasm (reviewed indeFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated byreference in its entirety). Two polymer approaches that have yieldedrobust in vivo delivery of nucleic acids, in this case with smallinterfering RNA (siRNA), are dynamic polyconjugates andcyclodextrin-based nanoparticles. The first of these delivery approachesuses dynamic polyconjugates and has been shown in vivo in mice toeffectively deliver siRNA and silence endogenous target mRNA inhepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887; herein incorporated by reference in its entirety). Thisparticular approach is a multicomponent polymer system whose keyfeatures include a membrane-active polymer to which nucleic acid, inthis case siRNA, is covalently coupled via a disulfide bond and whereboth PEG (for charge masking) and N-acetylgalactosamine (for hepatocytetargeting) groups are linked via pH-sensitive bonds (Rozema et al., ProcNatl Acad Sci USA. 2007 104:12982-12887; herein incorporated byreference in its entirety). On binding to the hepatocyte and entry intothe endosome, the polymer complex disassembles in the low-pHenvironment, with the polymer exposing its positive charge, leading toendosomal escape and cytoplasmic release of the siRNA from the polymer.Through replacement of the N-acetylgalactosamine group with a mannosegroup, it was shown one could alter targeting from asialoglycoproteinreceptor-expressing hepatocytes to sinusoidal endothelium and Kupffercells. Another polymer approach involves using transferrin-targetedcyclodextrin-containing polycation nanoparticles. These nanoparticleshave demonstrated targeted silencing of the EWS-FLI1 gene product intransferrin receptor-expressing Ewing's sarcoma tumor cells(Hu-Lieskovan et al., Cancer Res. 2005 65: 8984-8982; hereinincorporated by reference in its entirety) and siRNA formulated in thesenanoparticles was well tolerated in non-human primates (Heidel et al.,Proc Natl Acad Sci USA 2007 104:5715-21; herein incorporated byreference in its entirety). Both of these delivery strategiesincorporate rational approaches using both targeted delivery andendosomal escape mechanisms.

The polymer formulation can permit the sustained or delayed release ofpolynucleotide, primary construct, or mmRNA (e.g., followingintramuscular or subcutaneous injection). The altered release profilefor the polynucleotide, primary construct, or mmRNA can result in, forexample, translation of an encoded protein over an extended period oftime. The polymer formulation may also be used to increase the stabilityof the polynucleotide, primary construct, or mmRNA. Biodegradablepolymers have been previously used to protect nucleic acids other thanmmRNA from degradation and been shown to result in sustained release ofpayloads in vivo (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887; Sullivan et al., Expert Opin Drug Deliv. 20107:1433-1446; Convertine et al., Biomacromolecules. 2010 Oct. 1; Chu etal., Acc Chem. Res. 2012 Jan 13; Manganiello et al., Biomaterials. 201233:2301-2309; Benoit et al., Biomacromolecules. 2011 12:2708-2714;Singha et al., Nucleic Acid Ther. 2011 2:133-147; deFougerolles Hum GeneTher. 2008 19:125-132; Schaffert and Wagner, Gene Ther. 200816:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 20118:1455-1468; Davis, Mol. Pharm. 2009 6:659-668; Davis, Nature 2010464:1067-1070; each of which is herein incorporated by reference in itsentirety).

In one embodiment, the pharmaceutical compositions may be sustainedrelease formulations. In a further embodiment, the sustained releaseformulations may be for subcutaneous delivery. Sustained releaseformulations may include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.),surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,Ga.), TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

As a non-limiting example modified mRNA may be formulated in PLGAmicrospheres by preparing the PLGA microspheres with tunable releaserates (e.g., days and weeks) and encapsulating the modified mRNA in thePLGA microspheres while maintaining the integrity of the modified mRNAduring the encapsulation process. EVAc are non-biodegradeable,biocompatible polymers which are used extensively in pre-clinicalsustained release implant applications (e.g., extended release productsOcusert a pilocarpine ophthalmic insert for glaucoma or progestasert asustained release progesterone intrauterine device; transdermal deliverysystems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407NF is a hydrophilic, non-ionic surfactant triblock copolymer ofpolyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosityat temperatures less than 5° C. and forms a solid gel at temperaturesgreater than 15° C. PEG-based surgical sealants comprise two syntheticPEG components mixed in a delivery device which can be prepared in oneminute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE®and natural polymers are capable of in-situ gelation at the site ofadministration. They have been shown to interact with protein andpeptide therapeutic candidates through ionic ineraction to provide astabilizing effect.

Polymer formulations can also be selectively targeted through expressionof different ligands as exemplified by, but not limited by, folate,transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad SciUSA. 2007 104:12982-12887; Davis, Mol. Pharm. 2009 6:659-668; Davis,Nature 2010 464:1067-1070; each of which is herein incorporated byreference in its entirety).

The modified nucleic acid, and mmRNA of the invention may be formulatedwith or in a polymeric compound. The polymer may include at least onepolymer such as, but not limited to, polyethenes, polyethylene glycol(PEG), poly(1-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer,biodegradable cationic lipopolymer, polyethyleneimine (PEI),cross-linked branched poly(alkylene imines), a polyamine derivative, amodified poloxamer, a biodegradable polymer, elastic biodegradablepolymer, biodegradable block copolymer, biodegradable random copolymer,biodegradable polyester copolymer, biodegradable polyester blockcopolymer, biodegradable polyester block random copolymer, multiblockcopolymers, linear biodegradable copolymer,poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradablecross-linked cationic multi-block copolymers, polycarbonates,polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), acrylic polymers, amine-containingpolymers, dextran polymers, dextran polymer derivatives or combinationsthereof.

As a non-limiting example, the modified nucleic acid or mmRNA of theinvention may be formulated with the polymeric compound of PEG graftedwith PLL as described in U.S. Pat. No. 6,177,274; herein incorporated byreference in its entirety. The formulation may be used for transfectingcells in vitro or for in vivo delivery of the modified nucleic acid andmmRNA. In another example, the modified nucleic acid and mmRNA may besuspended in a solution or medium with a cationic polymer, in a drypharmaceutical composition or in a solution that is capable of beingdried as described in U.S. Pub. Nos. 20090042829 and 20090042825; eachof which are herein incorporated by reference in their entireties.

As another non-limiting example the polynucleotides, primary constructsor mmRNA of the invention may be formulated with a PLGA-PEG blockcopolymer (see US Pub. No. US20120004293 and U.S. Pat. No. 8,236,330,herein incorporated by reference in their entireties) or PLGA-PEG-PLGAblock copolymers (See U.S. Pat. No. 6,004,573, herein incorporated byreference in its entirety). As a non-limiting example, thepolynucleotides, primary constructs or mmRNA of the invention may beformulated with a diblock copolymer of PEG and PLA or PEG and PLGA (seeUS Pat No 8,246,968, herein incorporated by reference in its entirety).

A polyamine derivative may be used to deliver nucleic acids or to treatand/or prevent a disease or to be included in an implantable orinjectable device (U.S. Pub. No. 20100260817 herein incorporated byreference in its entirety). As a non-limiting example, a pharmaceuticalcomposition may include the modified nucleic acids and mmRNA and thepolyamine derivative described in U.S. Pub. No. 20100260817 (thecontents of which are incorporated herein by reference in its entirety.As a non-limiting example the polynucleotides, primary constructs andmmRNA of the present invention may be delivered using a polyamindepolymer such as, but not limited to, a polymer comprising a 1,3-dipolaraddition polymer prepared by combining a carbohydrate diazide monomerwith a dilkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280;herein incorporated by reference in its entirety).

In one embodiment, the polynucleotides, primary constructs or mmRNA ofthe present invention may be formulated with at least one polymer and/orderivatives thereof described in International Publication Nos.WO2011115862, WO2012082574 and WO2012068187 and U.S. Pub. No.20120283427, each of which are herein incorporated by reference in theirentireties. In another embodiment, the modified nucleic acid or mmRNA ofthe present invention may be formulated with a polymer of formula Z asdescribed in WO2011115862, herein incorporated by reference in itsentirety. In yet another embodiment, the modified nucleic acid or mmRNAmay be formulated with a polymer of formula Z, Z′ or Z″ as described inInternational Pub. Nos. WO2012082574 or WO2012068187 and U.S. Pub. No.2012028342, each of which are herein incorporated by reference in theirentireties. The polymers formulated with the modified RNA of the presentinvention may be synthesized by the methods described in InternationalPub. Nos. WO2012082574 or WO2012068187, each of which are hereinincorporated by reference in their entireties.

The polynucleotides, primary constructs or mmRNA of the invention may beformulated with at least one acrylic polymer. Acrylic polymers includebut are not limited to, acrylic acid, methacrylic acid, acrylic acid andmethacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethylmethacrylates, cyanoethyl methacrylate, amino alkyl methacrylatecopolymer, poly(acrylic acid), poly(methacrylic acid),polycyanoacrylates and combinations thereof.

Formulations of polynucleotides, primary constructs or mmRNA of theinvention may include at least one amine-containing polymer such as, butnot limited to polylysine, polyethylene imine, poly(amidoamine)dendrimers or combinations thereof.

For example, the modified nucleic acid or mmRNA of the invention may beformulated in a pharmaceutical compound including a poly(alkyleneimine), a biodegradable cationic lipopolymer, a biodegradable blockcopolymer, a biodegradable polymer, or a biodegradable random copolymer,a biodegradable polyester block copolymer, a biodegradable polyesterpolymer, a biodegradable polyester random copolymer, a linearbiodegradable copolymer, PAGA, a biodegradable cross-linked cationicmulti-block copolymer or combinations thereof. The biodegradablecationic lipopolymer may be made by methods known in the art and/ordescribed in U.S. Pat. No. 6,696,038, U.S. App. Nos. 20030073619 and20040142474 each of which is herein incorporated by reference in theirentireties. The poly(alkylene imine) may be made using methods known inthe art and/or as described in U.S. Pub. No. 20100004315, hereinincorporated by reference in its entirety. The biodegradabale polymer,biodegradable block copolymer, the biodegradable random copolymer,biodegradable polyester block copolymer, biodegradable polyesterpolymer, or biodegradable polyester random copolymer may be made usingmethods known in the art and/or as described in U.S. Pat. Nos. 6,517,869and 6,267,987, the contents of which are each incorporated herein byreference in their entirety. The linear biodegradable copolymer may bemade using methods known in the art and/or as described in U.S. Pat. No.6,652,886. The PAGA polymer may be made using methods known in the artand/or as described in U.S. Pat. No. 6,217,912 herein incorporated byreference in its entirety. The PAGA polymer may be copolymerized to forma copolymer or block copolymer with polymers such as but not limited to,poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines,polylactides and poly(lactide-co-glycolides). The biodegradablecross-linked cationic multi-block copolymers may be made my methodsknown in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S.Pub. No. 2012009145 each of which are herein incorporated by referencein their entireties. For example, the multi-block copolymers may besynthesized using linear polyethyleneimine (LPEI) blocks which havedistinct patterns as compared to branched polyethyleneimines. Further,the composition or pharmaceutical composition may be made by the methodsknown in the art, described herein, or as described in U.S. Pub. No.20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912 each of which areherein incorporated by reference in their entireties.

The polynucleotides, primary constructs, and mmRNA of the invention maybe formulated with at least one degradable polyester which may containpolycationic side chains. Degradeable polyesters include, but are notlimited to, poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester), and combinations thereof. In anotherembodiment, the degradable polyesters may include a PEG conjugation toform a PEGylated polymer.

The polynucleotides, primary construct, mmRNA of the invention may beformulated with at least one crosslinkable polyester. Crosslinkablepolyesters include those known in the art and described in US Pub. No.20120269761, herein incorporated by reference in its entirety.

In one embodiment, the polymers described herein may be conjugated to alipid-terminating PEG. As a non-limiting example, PLGA may be conjugatedto a lipid-terminating PEG forming PLGA-DSPE-PEG. As anothernon-limiting example, PEG conjugates for use with the present inventionare described in International Publication No. WO2008103276, hereinincorporated by reference in its entirety. The polymers may beconjugated using a ligand conjugate such as, but not limited to, theconjugates described in U.S. Pat. No. 8,273,363, herein incorporated byreference in its entirety.

In one embodiment, the modified RNA described herein may be conjugatedwith another compound. Non-limiting examples of conjugates are describedin U.S. Pat. Nos. 7,964,578 and 7,833,992, each of which are hereinincorporated by reference in their entireties. In another embodiment,modified RNA of the present invention may be conjugated with conjugatesof formula 1-122 as described in U.S. Pat. Nos. 7,964,578 and 7,833,992,each of which are herein incorporated by reference in their entireties.The polynucleotides, primary constructs and/or mmRNA described hereinmay be conjugated with a metal such as, but not limited to, gold. (Seee.g., Giljohann et al. Journ. Amer. Chem. Soc. 2009 131(6): 2072-2073;herein incorporated by reference in its entirety). In anotherembodiment, the polynucleotides, primary constructs and/or mmRNAdescribed herein may be conjugated and/or encapsulated ingold-nanoparticles. (Interantional Pub. No. WO201216269 and U.S. Pub.No. 20120302940; each of which is herein incorporated by reference inits entirety).

As described in U.S. Pub. No. 20100004313, herein incorporated byreference in its entirety, a gene delivery composition may include anucleotide sequence and a poloxamer. For example, the modified nucleicacid and mmRNA of the present inveition may be used in a gene deliverycomposition with the poloxamer described in U.S. Pub. No. 20100004313.

In one embodiment, the polymer formulation of the present invention maybe stabilized by contacting the polymer formulation, which may include acationic carrier, with a cationic lipopolymer which may be covalentlylinked to cholesterol and polyethylene glycol groups. The polymerformulation may be contacted with a cationic lipopolymer using themethods described in U.S. Pub. No. 20090042829 herein incorporated byreference in its entirety. The cationic carrier may include, but is notlimited to, polyethylenimine, poly(trimethylenimine),poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine,dideoxy-diamino-b-cyclodextrin, spermine, spermidine,poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine),poly(arginine), cationized gelatin, dendrimers, chitosan,1,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),3B-[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride(DC-Cholesterol HCl) diheptadecylamidoglycyl spermidine (DOGS),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride DODAC) andcombinations thereof.

The polynucleotides, primary constructs and/or mmRNA of the inventionmay be formulated in a polyplex of one or more polymers (U.S. Pub. No.20120237565 and 20120270927; each of which is herein incorporated byreference in its entirety). In one embodiment, the polyplex comprisestwo or more cationic polymers. The catioinic polymer may comprise apoly(ethylene imine) (PEI) such as linear PEI.

The polynucleotide, primary construct, and mmRNA of the invention canalso be formulated as a nanoparticle using a combination of polymers,lipids, and/or other biodegradable agents, such as, but not limited to,calcium phosphate. Components may be combined in a core-shell, hybrid,and/or layer-by-layer architecture, to allow for fine-tuning of thenanoparticle so to delivery of the polynucleotide, primary construct andmmRNA may be enhanced (Wang et al., Nat. Mater. 2006 5:791-796; Fulleret al., Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv Drug DelivRev. 2011 63:748-761; Endres et al., Biomaterials. 2011 32:7721-7731; Suet al., Mol. Pharm. 2011 Jun. 6; 8(3):774-87; herein incorporated byreference in its entirety). As a non-limiting example, the nanoparticlemay comprise a plurality of polymers such as, but not limited tohydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers(e.g., PEG) and/or hydrophilic polymers (International Pub. No.WO20120225129; herein incorporated by reference in its entirety).

Biodegradable calcium phosphate nanoparticles in combination with lipidsand/or polymers have been shown to deliver polynucleotides, primaryconstructs and mmRNA in vivo. In one embodiment, a lipid coated calciumphosphate nanoparticle, which may also contain a targeting ligand suchas anisamide, may be used to deliver the polynucleotide, primaryconstruct and mmRNA of the present invention. For example, toeffectively deliver siRNA in a mouse metastatic lung model a lipidcoated calcium phosphate nanoparticle was used (Li et al., J Contr Rel.2010 142: 416-421; Li et al., J Contr Rel. 2012 158:108-114; Yang etal., Mol. Ther. 2012 20:609-615; herein incorporated by reference in itsentirety). This delivery system combines both a targeted nanoparticleand a component to enhance the endosomal escape, calcium phosphate, inorder to improve delivery of the siRNA.

In one embodiment, calcium phosphate with a PEG-polyanion blockcopolymer may be used to delivery polynucleotides, primary constructsand mmRNA (Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa etal., J Contr Rel. 2006 111:368-370; herein incorporated by reference inits entirety).

In one embodiment, a PEG-charge-conversional polymer (Pitella et al.,Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle todeliver the polynucleotides, primary constructs and mmRNA of the presentinvention. The PEG-charge-conversional polymer may improve upon thePEG-polyanion block copolymers by being cleaved into a polycation atacidic pH, thus enhancing endosomal escape.

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-13001). The complexation, delivery, and internalization of thepolymeric nanoparticles can be precisely controlled by altering thechemical composition in both the core and shell components of thenanoparticle. For example, the core-shell nanoparticles may efficientlydeliver siRNA to mouse hepatocytes after they covalently attachcholesterol to the nanoparticle.

In one embodiment, a hollow lipid core comprising a middle PLGA layerand an outer neutral lipid layer containing PEG may be used to deliveryof the polynucleotide, primary construct and mmRNA of the presentinvention. As a non-limiting example, in mice bearing aluciferease-expressing tumor, it was determined that thelipid-polymer-lipid hybrid nanoparticle significantly suppressedluciferase expression, as compared to a conventional lipoplex (Shi etal, Angew Chem Int Ed. 2011 50:7027-7031; herein incorporated byreference in its entirety).

In one embodiment, the lipid nanoparticles may comprise a core of themodified nucleic acid molecules disclosed herein and a polymer shell.The polymer shell may be any of the polymers described herein and areknown in the art. In an additional embodiment, the polymer shell may beused to protect the modified nucleic acids in the core.

Core-shell nanoparticles for use with the modified nucleic acidmolecules of the present invention are described and may be formed bythe methods described in U.S. Pat. No. 8,313,777 herein incorporated byreference in its entirety.

In one embodiment, the core-shell nanoparticles may comprise a core ofthe modified nucleic acid molecules disclosed herein and a polymershell. The polymer shell may be any of the polymers described herein andare known in the art. In an additional embodiment, the polymer shell maybe used to protect the modified nucleic acid molecules in the core. As anon-limiting example, the core-shell nanoparticle may be used to treatan eye disease or disorder (See e.g. US Publication No. 20120321719,herein incorporated by reference in its entirety).

In one embodiment, the polymer used with the formulations describedherein may be a modified polymer (such as, but not limited to, amodified polyacetal) as described in International Publication No.WO2011120053, herein incorporated by reference in its entirety.

Peptides and Proteins

The polynucleotide, primary construct, and mmRNA of the invention can beformulated with peptides and/or proteins in order to increasetransfection of cells by the polynucleotide, primary construct, ormmRNA. In one embodiment, peptides such as, but not limited to, cellpenetrating peptides and proteins and peptides that enable intracellulardelivery may be used to deliver pharmaceutical formulations. Anon-limiting example of a cell penetrating peptide which may be usedwith the pharmaceutical formulations of the present invention includes acell-penetrating peptide sequence attached to polycations thatfacilitates delivery to the intracellular space, e.g., HIV-derived TATpeptide, penetratins, transportans, or hCT derived cell-penetratingpeptides (see, e.g., Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel,Cell-Penetrating Peptides: Processes and Applications (CRC Press, BocaRaton Fla., 2002); E1-Andaloussi et al., Curr. Pharm. Des.11(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life. Sci.62(16):1839-49 (2005), all of which are incorporated herein by referencein their entirety). The compositions can also be formulated to include acell penetrating agent, e.g., liposomes, which enhance delivery of thecompositions to the intracellular space. Polynucleotides, primaryconstructs, and mmRNA of the invention may be complexed to peptidesand/or proteins such as, but not limited to, peptides and/or proteinsfrom Aileron Therapeutics (Cambridge, Mass.) and Permeon Biologics(Cambridge, Mass.) in order to enable intracellular delivery (Cronicanet al., ACS Chem. Biol. 2010 5:747-752; McNaughton et al., Proc. Natl.Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug Des. 200973:3-6; Verdine and Hilinski, Methods Enzymol. 2012; 503:3-33; all ofwhich are herein incorporated by reference in its entirety).

In one embodiment, the cell-penetrating polypeptide may comprise a firstdomain and a second domain. The first domain may comprise a superchargedpolypeptide. The second domain may comprise a protein-binding partner.As used herein, “protein-binding partner” includes, but are not limitedto, antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further comprise anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where the polynucleotide, primary construct, or mmRNA may beintroduced.

Formulations of the including peptides or proteins may be used toincrease cell transfection by the polynucleotide, primary construct, ormmRNA, alter the biodistribution of the polynucleotide, primaryconstruct, or mmRNA (e.g., by targeting specific tissues or cell types),and/or increase the translation of encoded protein. (See e.g.,International Pub. No. WO2012110636; herein incorporated by reference inits entirety).

Cells

The polynucleotide, primary construct, and mmRNA of the invention can betransfected ex vivo into cells, which are subsequently transplanted intoa subject. As non-limiting examples, the pharmaceutical compositions mayinclude red blood cells to deliver modified RNA to liver and myeloidcells, virosomes to deliver modified RNA in virus-like particles (VLPs),and electroporated cells such as, but not limited to, from MAXCYTE®(Gaithersburg, Md.) and from ERYTECH® (Lyon, France) to deliver modifiedRNA. Examples of use of red blood cells, viral particles andelectroporated cells to deliver payloads other than mmRNA have beendocumented (Godfrin et al., Expert Opin Biol Ther. 2012 12:127-133; Fanget al., Expert Opin Biol Ther. 2012 12:385-389; Hu et al., Proc NatlAcad Sci USA. 2011 108:10980-10985; Lund et al., Pharm Res. 201027:400-420; Huckriede et al., J Liposome Res. 2007; 17:39-47; Cusi, HumVaccin. 2006 2:1-7; de Jonge et al., Gene Ther. 2006 13:400-411; all ofwhich are herein incorporated by reference in its entirety).

The polynucleotides, primary constructs and mmRNA may be delivered insynthetic VLPs synthesized by the methods described in International PubNo. WO2011085231 and US Pub No. 20110171248, each of which are hereinincorporated by reference in their entireties.

Cell-based formulations of the polynucleotide, primary construct, andmmRNA of the invention may be used to ensure cell transfection (e.g., inthe cellular carrier), alter the biodistribution of the polynucleotide,primary construct, or mmRNA (e.g., by targeting the cell carrier tospecific tissues or cell types), and/or increase the translation ofencoded protein.

A variety of methods are known in the art and suitable for introductionof nucleic acid into a cell, including viral and non-viral mediatedtechniques. Examples of typical non-viral mediated techniques include,but are not limited to, electroporation, calcium phosphate mediatedtransfer, nucleofection, sonoporation, heat shock, magnetofection,liposome mediated transfer, microinjection, microprojectile mediatedtransfer (nanoparticles), cationic polymer mediated transfer(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like)or cell fusion.

The technique of sonoporation, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofthe cell plasma membrane. Sonoporation methods are known to those in theart and are used to deliver nucleic acids in vivo (Yoon and Park, ExpertOpin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr PharmBiotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 200714:465-475; all herein incorporated by reference in their entirety).Sonoporation methods are known in the art and are also taught forexample as it relates to bacteria in US Patent Publication 20100196983and as it relates to other cell types in, for example, US PatentPublication 20100009424, each of which are incorporated herein byreference in their entirety.

Electroporation techniques are also well known in the art and are usedto deliver nucleic acids in vivo and clinically (Andre et al., Curr GeneTher. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 201010:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all hereinincorporated by reference in their entirety). In one embodiment,polynucleotides, primary constructs or mmRNA may be delivered byelectroporation as described in Example 8.

Hyaluronidase

The intramuscular or subcutaneous localized injection of polynucleotide,primary construct, or mmRNA of the invention can include hyaluronidase,which catalyzes the hydrolysis of hyaluronan. By catalyzing thehydrolysis of hyaluronan, a constituent of the interstitial barrier,hyaluronidase lowers the viscosity of hyaluronan, thereby increasingtissue permeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440;herein incorporated by reference in its entirety). It is useful to speedtheir dispersion and systemic distribution of encoded proteins producedby transfected cells. Alternatively, the hyaluronidase can be used toincrease the number of cells exposed to a polynucleotide, primaryconstruct, or mmRNA of the invention administered intramuscularly orsubcutaneously.

Nanoparticle Mimics

The polynucleotide, primary construct or mmRNA of the invention may beencapsulated within and/or absorbed to a nanoparticle mimic. Ananoparticle mimic can mimic the delivery function organisms orparticles such as, but not limited to, pathogens, viruses, bacteria,fungus, parasites, prions and cells. As a non-limiting example thepolynucleotide, primary construct or mmRNA of the invention may beencapsulated in a non-viron particle which can mimic the deliveryfunction of a virus (see International Pub. No. WO2012006376 hereinincorporated by reference in its entirety).

Nanotubes

The polynucleotides, primary constructs or mmRNA of the invention can beattached or otherwise bound to at least one nanotube such as, but notlimited to, rosette nanotubes, rosette nanotubes having twin bases witha linker, carbon nanotubes and/or single-walled carbon nanotubes, Thepolynucleotides, primary constructs or mmRNA may be bound to thenanotubes through forces such as, but not limited to, steric, ionic,covalent and/or other forces.

In one embodiment, the nanotube can release one or more polynucleotides,primary constructs or mmRNA into cells. The size and/or the surfacestructure of at least one nanotube may be altered so as to govern theinteraction of the nanotubes within the body and/or to attach or bind tothe polynucleotides, primary constructs or mmRNA disclosed herein. Inone embodiment, the building block and/or the functional groups attachedto the building block of the at least one nanotube may be altered toadjust the dimensions and/or properties of the nanotube. As anon-limiting example, the length of the nanotubes may be altered tohinder the nanotubes from passing through the holes in the walls ofnormal blood vessels but still small enough to pass through the largerholes in the blood vessels of tumor tissue.

In one embodiment, at least one nanotube may also be coated withdelivery enhancing compounds including polymers, such as, but notlimited to, polyethylene glycol. In another embodiment, at least onenanotube and/or the polynucleotides, primary constructs or mmRNA may bemixed with pharmaceutically acceptable excipients and/or deliveryvehicles.

In one embodiment, the polynucleotides, primary constructs or mmRNA areattached and/or otherwise bound to at least one rosette nanotube. Therosette nanotubes may be formed by a process known in the art and/or bythe process described in International Publication No. WO2012094304,herein incorporated by reference in its entirety. At least onepolynucleotide, primary construct and/or mmRNA may be attached and/orotherwise bound to at least one rosette nanotube by a process asdescribed in International Publication No. WO2012094304, hereinincorporated by reference in its entirety, where rosette nanotubes ormodules forming rosette nanotubes are mixed in aqueous media with atleast one polynucleotide, primary construct and/or mmRNA underconditions which may cause at least one polynucleotide, primaryconstruct or mmRNA to attach or otherwise bind to the rosette nanotubes.

In one embodiment, the polynucleotides, primary constructs or mmRNA maybe attached to and/or otherwise bound to at least one carbon nanotube.As a non-limiting example, the polynucleotides, primary constructs ormmRNA may be bound to a linking agent and the linked agent may be boundto the carbon nanotube (See e.g., U.S. Pat. No. 8,246,995; hereinincorporated by reference in its entirety). The carbon nanotube may be asingle-walled nanotube (See e.g., U.S. Pat. No. 8,246,995; hereinincorporated by reference in its entirety).

Conjugates

The polynucleotides, primary constructs, and mmRNA of the inventioninclude conjugates, such as a polynucleotide, primary construct, ormmRNA covalently linked to a carrier or targeting group, or includingtwo encoding regions that together produce a fusion protein (e.g.,bearing a targeting group and therapeutic protein or peptide).

The conjugates of the invention include a naturally occurring substance,such as a protein (e.g., human serum albumin (HSA), low-densitylipoprotein (LDL), high-density lipoprotein (HDL), or globulin); ancarbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin,cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examplesof polyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Representative U.S. patents that teach the preparation of polynucleotideconjugates, particularly to RNA, include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664;6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which isherein incorporated by reference in their entireties.

In one embodiment, the conjugate of the present invention may functionas a carrier for the modified nucleic acids and mmRNA of the presentinvention. The conjugate may comprise a cationic polymer such as, butnot limited to, polyamine, polylysine, polyalkylenimine, andpolyethylenimine which may be grafted to with poly(ethylene glycol). Asa non-limiting example, the conjugate may be similar to the polymericconjugate and the method of synthesizing the polymeric conjugatedescribed in U.S. Pat. No. 6,586,524 herein incorporated by reference inits entirety.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell. Targeting groups mayalso include hormones and hormone receptors. They can also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,multivalent fucose, or aptamers. The ligand can be, for example, alipopolysaccharide, or an activator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, GalNAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein.

In one embodiment, pharmaceutical compositions of the present inventionmay include chemical modifications such as, but not limited to,modifications similar to locked nucleic acids.

Representative U.S. Patents that teach the preparation of locked nucleicacid (LNA) such as those from Santaris, include, but are not limited to,the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is hereinincorporated by reference in its entirety.

Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found, for example, in Nielsenet al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include polynucleotides,primary constructs or mmRNA with phosphorothioate backbones andoligonucleosides with other modified backbones, and in particular—CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) orMMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone isrepresented as —O—P(O)₂—O—CH₂—] of the above-referenced U.S. Pat. No.5,489,677, and the amide backbones of the above-referenced U.S. Pat. No.5,602,240. In some embodiments, the polynucletotides featured hereinhave morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modifications at the 2′ position may also aid in delivery. Preferably,modifications at the 2′ position are not located in a polypeptide-codingsequence, i.e., not in a translatable region. Modifications at the 2′position may be located in a 5′UTR, a 3′UTR and/or a tailing region.Modifications at the 2′ position can include one of the following at the2′ position: H (i.e., 2′-deoxy); F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, the polynucleotides,primary constructs or mmRNA include one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties, or a group for improving the pharmacodynamicproperties, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-0—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below. Othermodifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions, particularly the 3′ position of the sugar onthe 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ positionof 5′ terminal nucleotide. Polynucleotides of the invention may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920 and each of which isherein incorporated by reference.

In still other embodiments, the polynucleotide, primary construct, ormmRNA is covalently conjugated to a cell penetrating polypeptide. Thecell-penetrating peptide may also include a signal sequence. Theconjugates of the invention can be designed to have increased stability;increased cell transfection; and/or altered the biodistribution (e.g.,targeted to specific tissues or cell types).

In one embodiment, the polynucleotides, primary constructs or mmRNA maybe conjugated to an agent to enhance delivery. As a non-limitingexample, the agent may be a monomer or polymer such as a targetingmonomer or a polymer having targeting blocks as described inInternational Publication No. WO2011062965, herein incorporated byreference in its entirety. In another non-limiting example, the agentmay be a transport agent covalently coupled to the polynucleotides,primary constructs or mmRNA of the present invention (See e.g., U.S.Pat. Nos. 6,835,393 and 7,374,778, each of which is herein incorporatedby reference in its entirety). In yet another non-limiting example, theagent may be a membrane barrier transport enhancing agent such as thosedescribed in U.S. Pat. Nos. 7,737,108 and 8,003,129, each of which isherein incorporated by reference in its entirety.

In another embodiment, polynucleotides, primary constructs or mmRNA maybe conjugated to SMARTT POLYMER TECHNOLOGY® (PHASERX®, Inc. Seattle,Wash.).

Self-Assembled Nanoparticles Nucleic Acid Self-Assembled Nanoparticles

Self-assembled nanoparticles have a well-defined size which may beprecisely controlled as the nucleic acid strands may be easilyreprogrammable. For example, the optimal particle size for acancer-targeting nanodelivery carrier is 20-100 nm as a diameter greaterthan 20 nm avoids renal clearance and enhances delivery to certaintumors through enhanced permeability and retention effect. Usingself-assembled nucleic acid nanoparticles a single uniform population insize and shape having a precisely controlled spatial orientation anddensity of cancer-targeting ligands for enhanced delivery. As anon-limiting example, oligonucleotide nanoparticles were prepared usingprogrammable self-assembly of short DNA fragments and therapeuticsiRNAs. These nanoparticles are molecularly identical with controllableparticle size and target ligand location and density. The DNA fragmentsand siRNAs self-assembled into a one-step reaction to generate DNA/siRNAtetrahedral nanoparticles for targeted in vivo delivery. (Lee et al.,Nature Nanotechnology 2012 7:389-393; herein incorporated by referencein its entirety).

In one embodiment, the polynucleotides, primary constructs and/or mmRNAdisclosed herein may be formulated as self-assembled nanoparticles. As anon-limiting example, nucleic acids may be used to make nanoparticleswhich may be used in a delivery system for the polynucleotides, primaryconstructs and/or mmRNA of the present invention (See e.g.,International Pub. No. WO2012125987; herein incorporated by reference inits entirety).

In one embodiment, the nucleic acid self-assembled nanoparticles maycomprise a core of the polynucleotides, primary constructs or mmRNAdisclosed herein and a polymer shell. The polymer shell may be any ofthe polymers described herein and are known in the art. In an additionalembodiment, the polymer shell may be used to protect thepolynucleotides, primary contructs and mmRNA in the core.

Polymer-Based Self-Assembled Nanoparticles

Polymers may be used to form sheets which self-assembled intonanoparticles. These nanoparticles may be used to deliver thepolynucleotides, primary constructs and mmRNA of the present invention.In one embodiment, these self-assembled nanoparticles may bemicrosponges formed of long polymers of RNA hairpins which form intocrystalline ‘pleated’ sheets before self-assembling into microsponges.These microsponges are densely-packed sponge like microparticles whichmay function as an efficient carrier and may be able to deliver cargo toa cell. The microsponges may be from 1 um to 300 nm in diameter. Themicrosponges may be complexed with other agents known in the art to formlarger microsponges. As a non-limiting example, the microsponge may becomplexed with an agent to form an outer layer to promote cellularuptake such as polycation polyethyleneime (PEI). This complex can form a250-nm diameter particle that can remain stable at high temperatures(150° C.) (Grabow and Jaegar, Nature Materials 2012, 11:269-269; hereinincorporated by reference in its entirety). Additionally thesemicrosponges may be able to exhibit an extraordinary degree ofprotection from degradation by ribonucleases.

In another embodiment, the polymer-based self-assembled nanoparticlessuch as, but not limited to, microsponges, may be fully programmablenanoparticles. The geometry, size and stoichiometry of the nanoparticlemay be precisely controlled to create the optimal nanoparticle fordelivery of cargo such as, but not limited to, polynucleotides, primaryconstructs and/or mmRNA.

In one embodiment, the polymer based nanoparticles may comprise a coreof the polynucleotides, primary constructs and/or mmRNA disclosed hereinand a polymer shell. The polymer shell may be any of the polymersdescribed herein and are known in the art. In an additional embodiment,the polymer shell may be used to protect the polynucleotides, primaryconstruct and/or mmRNA in the core.

In yet another embodiment, the polymer based nanoparticle may comprise anon-nucleic acid polymer comprising a plurality of heterogenous monomerssuch as those described in Interantional Publication No. WO2013009736,herein incorporated by reference in its entirety.

Inorganic Nanoparticles

The polynucleotides, primary constructs and/or mmRNAs of the presentinvention may be formulated in inorganic nanoparticles (U.S. Pat. No.8,257,745, herein incorporated by reference in its entirety). Theinorganic nanoparticles may include, but are not limited to, claysubstances that are water swellable. As a non-limiting example, theinorganic nanoparticle may include synthetic smectite clays which aremade from simple silicates (See e.g., U.S. Pat. Nos. 5,585,108 and8,257,745 each of which are herein incorporated by reference in theirentirety).

In one embodiment, the inorganic nanoparticles may comprise a core ofthe modified nucleic acids disclosed herein and a polymer shell. Thepolymer shell may be any of the polymers described herein and are knownin the art. In an additional embodiment, the polymer shell may be usedto protect the modified nucleic acids in the core.

Semi-conductive and Metallic Nanoparticles

The polynucleotides, primary constructs and/or mmRNAs of the presentinvention may be formulated in water-dispersible nanoparticle comprisinga semiconductive or metallic material (U.S. Pub. No. 20120228565; hereinincorporated by reference in its entirety) or formed in a magneticnanoparticle (U.S. Pub. No. 20120265001 and 20120283503; each of whichis herein incorporated by reference in its entirety). Thewater-dispersible nanoparticles may be hydrophobic nanoparticles orhydrophilic nanoparticles.

In one embodiment, the semi-conductive and/or metallic nanoparticles maycomprise a core of the polynucleotides, primary constructs and/or mmRNAdisclosed herein and a polymer shell. The polymer shell may be any ofthe polymers described herein and are known in the art. In an additionalembodiment, the polymer shell may be used to protect thepolynucleotides, primary constructs and/or mmRNA in the core.

Gels and Hydrogels

In one embodiment, the polynucleotides, primary constructs and/or mmRNAdisclosed herein may be encapsulated into any hydrogel known in the artwhich may form a gel when injected into a subject. Hydrogels are anetwork of polymer chains that are hydrophilic, and are sometimes foundas a colloidal gel in which water is the dispersion medium. Hydrogelsare highly absorbent (they can contain over 99% water) natural orsynthetic polymers. Hydrogels also possess a degree of flexibility verysimilar to natural tissue, due to their significant water content. Thehydrogel described herein may used to encapsulate lipid nanoparticleswhich are biocompatible, biodegradable and/or porous.

As a non-limiting example, the hydrogel may be an aptamer-functionalizedhydrogel. The aptamer-functionalized hydrogel may be programmed torelease one or more polynucleotides, primary constructs and/or mmRNAusing nucleic acid hybridization. (Battig et al., J. Am. Chem. Society.2012 134:12410-12413; herein incorporated by reference in its entirety).

As another non-limiting example, the hydrogel may be a shaped as aninverted opal.

The opal hydrogels exhibit higher swelling ratios and the swellingkinetics is an order of magnitude faster as well. Methods of producingopal hydrogels and description of opal hydrogels are described inInternational Pub. No. WO2012148684, herein incorporated by reference inits entirety.

In yet another non-limiting example, the hydrogel may be anantibacterial hydrogel. The antibacterial hydrogel may comprise apharmaceutical acceptable salt or organic material such as, but notlimited to pharmaceutical grade and/or medical grade silver salt andaloe vera gel or extract. (International Pub. No. WO2012151438, hereinincorporated by reference in its entirety).

In one embodiment, the modified mRNA may be encapsulated in a lipidnanoparticle and then the lipid nanoparticle may be encapsulated into ahyrdogel.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAdisclosed herein may be encapsulated into any gel known in the art. As anon-limiting example the gel may be a fluorouracil injectable gel or afluorouracil injectable gel containing a chemical compound and/or drugknown in the art. As another example, the polynucleotides, primaryconstructs and/or mmRNA may be encapsulated in a fluorouracil gelcontaining epinephrine (See e.g., Smith et al. Cancer Chemotherapty andPharmacology, 1999 44(4):267-274; herein incorporated by reference inits entirety).

In one embodiment, the polynucleotides, primary constructs and/or mmRNAdisclosed herein may be encapsulated into a fibrin gel, fibrin hydrogelor fibrin glue. In another embodiment, the polynucleotides, primaryconstructs and/or mmRNA may be formulated in a lipid nanoparticle or arapidly eliminated lipid nanoparticle prior to being encapsulated into afibrin gel, fibrin hydrogel or a fibrin glue. In yet another embodiment,the polynucleotides, primary constructs and/or mmRNA may be formulatedas a lipoplex prior to being encapsulated into a fibrin gel, hydrogel ora fibrin glue. Fibrin gels, hydrogels and glues comprise two components,a fibrinogen solution and a thrombin solution which is rich in calcium(See e.g., Spicer and Mikos, Journal of Controlled Release 2010. 148:49-55; Kidd et al. Journal of Controlled Release 2012. 157:80-85; eachof which is herein incorporated by reference in its entirety). Theconcentration of the components of the fibrin gel, hydrogel and/or gluecan be altered to change the characteristics, the network mesh size,and/or the degradation characteristics of the gel, hydrogel and/or gluesuch as, but not limited to changing the release characteristics of thefibrin gel, hydrogel and/or glue. (See e.g., Spicer and Mikos, Journalof Controlled Release 2010. 148: 49-55; Kidd et al. Journal ofControlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering2008. 14:119-128; each of which is herein incorporated by reference inits entirety). This feature may be advantageous when used to deliver themodified mRNA disclosed herein. (See e.g., Kidd et al. Journal ofControlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering2008. 14:119-128; each of which is herein incorporated by reference inits entirety).

Cations and Anions

Formulations of polynucleotides, primary constructs and/or mmRNAdisclosed herein may include cations or anions. In one embodiment, theformulations include metal cations such as, but not limited to, Zn2+,Ca2+, Cu2+, Mg+ and combinations thereof. As a non-limiting example,formulations may include polymers and a polynucleotides, primaryconstructs and/or mmRNA complexed with a metal cation (See e.g., U.S.Pat. Nos. 6,265,389 and 6,555,525, each of which is herein incorporatedby reference in its entirety).

Molded Nanoparticles and Microparticles

The polynucleotides, primary constructs and/or mmRNA disclosed hereinmay be formulated in nanoparticles and/or microparticles. Thesenanoparticles and/or microparticles may be molded into any size shapeand chemistry. As an example, the nanoparticles and/or microparticlesmay be made using the PRINT® technology by LIQUIDA TECHNOLOGIES®(Morrisville, N.C.) (See e.g., International Pub. No. WO2007024323;herein incorporated by reference in its entirety).

In one embodiment, the molded nanoparticles may comprise a core of thepolynucleotides, primary constructs and/or mmRNA disclosed herein and apolymer shell. The polymer shell may be any of the polymers describedherein and are known in the art. In an additional embodiment, thepolymer shell may be used to protect the polynucleotides, primaryconstruct and/or mmRNA in the core.

NanoJackets and NanoLiposomes

The polynucleotides, primary constructs and/or mmRNA disclosed hereinmay be formulated in NanoJackets and NanoLiposomes by Keystone Nano(State College, Pa.). NanoJackets are made of compounds that arenaturally found in the body including calcium, phosphate and may alsoinclude a small amount of silicates. Nanojackets may range in size from5 to 50 nm and may be used to deliver hydrophilic and hydrophobiccompounds such as, but not limited to, polynucleotides, primaryconstructs and/or mmRNA.

NanoLiposomes are made of lipids such as, but not limited to, lipidswhich naturally occur in the body. NanoLiposomes may range in size from60-80 nm and may be used to deliver hydrophilic and hydrophobiccompounds such as, but not limited to, polynucleotides, primaryconstructs and/or mmRNA. In one aspect, the polynucleotides, primaryconstructs and/or mmRNA disclosed herein are formulated in aNanoLiposome such as, but not limited to, Ceramide NanoLiposomes.

Excipients

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference in its entirety) disclosesvarious excipients used in formulating pharmaceutical compositions andknown techniques for the preparation thereof. Except insofar as anyconventional excipient medium is incompatible with a substance or itsderivatives, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical compositions.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEENn®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate[SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Delivery

The present disclosure encompasses the delivery of polynucleotides,primary constructs or mmRNA for any of therapeutic, pharmaceutical,diagnostic or imaging by any appropriate route taking into considerationlikely advances in the sciences of drug delivery. Delivery may be nakedor formulated.

Naked Delivery

The polynucleotides, primary constructs or mmRNA of the presentinvention may be delivered to a cell naked. As used herein in, “naked”refers to delivering polynucleotides, primary constructs or mmRNA freefrom agents which promote transfection. For example, thepolynucleotides, primary constructs or mmRNA delivered to the cell maycontain no modifications. The naked polynucleotides, primary constructsor mmRNA may be delivered to the cell using routes of administrationknown in the art and described herein.

Formulated Delivery

The polynucleotides, primary constructs or mmRNA of the presentinvention may be formulated, using the methods described herein. Theformulations may contain polynucleotides, primary constructs or mmRNAwhich may be modified and/or unmodified. The formulations may furtherinclude, but are not limited to, cell penetration agents, apharmaceutically acceptable carrier, a delivery agent, a bioerodible orbiocompatible polymer, a solvent, and a sustained-release deliverydepot. The formulated polynucleotides, primary constructs or mmRNA maybe delivered to the cell using routes of administration known in the artand described herein.

The compositions may also be formulated for direct delivery to an organor tissue in any of several ways in the art including, but not limitedto, direct soaking or bathing, via a catheter, by gels, powder,ointments, creams, gels, lotions, and/or drops, by using substrates suchas fabric or biodegradable materials coated or impregnated with thecompositions, and the like.

Administration

The polynucleotides, primary constructs or mmRNA of the presentinvention may be administered by any route which results in atherapeutically effective outcome. These include, but are not limited toenteral, gastroenteral, epidural, oral, transdermal, epidural(peridural), intracerebral (into the cerebrum), intracerebroventricular(into the cerebral ventricles), epicutaneous (application onto theskin), intradermal, (into the skin itself), subcutaneous (under theskin), nasal administration (through the nose), intravenous (into avein), intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection, (into the base of thepenis), intravaginal administration, intrauterine, extra-amnioticadministration, transdermal (diffusion through the intact skin forsystemic distribution), transmucosal (diffusion through a mucousmembrane), insufflation (snorting), sublingual, sublabial, enema, eyedrops (onto the conjunctiva), or in ear drops. In specific embodiments,compositions may be administered in a way which allows them cross theblood-brain barrier, vascular barrier, or other epithelial barrier.Non-limiting routes of administration for the polynucleotides, primaryconstructs or mmRNA of the present invention are described below.

Parenteral and Injectible Administration

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, oral compositions can include adjuvants such as wettingagents, emulsifying and suspending agents, sweetening, flavoring, and/orperfuming agents. In certain embodiments for parenteral administration,compositions are mixed with solubilizing agents such as CREMOPHOR,alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins,polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Rectal and Vaginal Administration

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Oral Administration

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups, and/or elixirs. In addition to active ingredients,liquid dosage forms may comprise inert diluents commonly used in the artsuch as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, oralcompositions can include adjuvants such as wetting agents, emulsifyingand suspending agents, sweetening, flavoring, and/or perfuming agents.In certain embodiments for parenteral administration, compositions aremixed with solubilizing agents such as CREMOPHOR®, alcohols, oils,modified oils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g. starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.glycerol), disintegrating agents (e.g. agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g. paraffin), absorptionaccelerators (e.g. quaternary ammonium compounds), wetting agents (e.g.cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin andbentonite clay), and lubricants (e.g. talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate), andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may comprise buffering agents.

Topical or Transdermal Administration

As described herein, compositions containing the polynucleotides,primary constructs or mmRNA of the invention may be formulated foradministration topically. The skin may be an ideal target site fordelivery as it is readily accessible. Gene expression may be restrictednot only to the skin, potentially avoiding nonspecific toxicity, butalso to specific layers and cell types within the skin.

The site of cutaneous expression of the delivered compositions willdepend on the route of nucleic acid delivery. Three routes are commonlyconsidered to deliver polynucleotides, primary constructs or mmRNA tothe skin: (i) topical application (e.g. for local/regional treatmentand/or cosmetic applications); (ii) intradermal injection (e.g. forlocal/regional treatment and/or cosmetic applications); and (iii)systemic delivery (e.g. for treatment of dermatologic diseases thataffect both cutaneous and extracutaneous regions). Polynucleotides,primary constructs or mmRNA can be delivered to the skin by severaldifferent approaches known in the art. Most topical delivery approacheshave been shown to work for delivery of DNA, such as but not limited to,topical application of non-cationic liposome—DNA complex, cationicliposome—DNA complex, particle-mediated (gene gun), puncture-mediatedgene transfections, and viral delivery approaches. After delivery of thenucleic acid, gene products have been detected in a number of differentskin cell types, including, but not limited to, basal keratinocytes,sebaceous gland cells, dermal fibroblasts and dermal macrophages.

In one embodiment, the invention provides for a variety of dressings(e.g., wound dressings) or bandages (e.g., adhesive bandages) forconveniently and/or effectively carrying out methods of the presentinvention. Typically dressing or bandages may comprise sufficientamounts of pharmaceutical compositions and/or polynucleotides, primaryconstructs or mmRNA described herein to allow a user to perform multipletreatments of a subject(s).

In one embodiment, the invention provides for the polynucleotides,primary constructs or mmRNA compositions to be delivered in more thanone injection.

In one embodiment, before topical and/or transdermal administration atleast one area of tissue, such as skin, may be subjected to a deviceand/or solution which may increase permeability. In one embodiment, thetissue may be subjected to an abrasion device to increase thepermeability of the skin (see U.S. Patent Publication No. 20080275468,herein incorporated by reference in its entirety). In anotherembodiment, the tissue may be subjected to an ultrasound enhancementdevice. An ultrasound enhancement device may include, but is not limitedto, the devices described in U.S. Publication No. 20040236268 and U.S.Pat. Nos. 6,491,657 and 6,234,990; each of which are herein incorporatedby reference in their entireties. Methods of enhancing the permeabilityof tissue are described in U.S. Publication Nos. 20040171980 and20040236268 and U.S. Pat. No. 6,190,315; each of which are hereinincorporated by reference in their entireties.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein.The permeability of skin may be measured by methods known in the artand/or described in U.S. Pat. No. 6,190,315, herein incorporated byreference in its entirety. As a non-limiting example, a modified mRNAformulation may be delivered by the drug delivery methods described inU.S. Pat. No. 6,190,315, herein incorporated by reference in itsentirety.

In another non-limiting example tissue may be treated with a eutecticmixture of local anesthetics (EMLA) cream before, during and/or afterthe tissue may be subjected to a device which may increase permeability.Katz et al. (Anesth Analg (2004); 98:371-76; herein incorporated byreference in its entirety) showed that using the EMLA cream incombination with a low energy, an onset of superficial cutaneousanalgesia was seen as fast as 5 minutes after a pretreatment with a lowenergy ultrasound.

In one embodiment, enhancers may be applied to the tissue before,during, and/or after the tissue has been treated to increasepermeability. Enhancers include, but are not limited to, transportenhancers, physical enhancers, and cavitation enhancers. Non-limitingexamples of enhancers are described in U.S. Pat. No. 6,190,315, hereinincorporated by reference in its entirety.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein,which may further contain a substance that invokes an immune response.In another non-limiting example, a formulation containing a substance toinvoke an immune response may be delivered by the methods described inU.S. Publication Nos. 20040171980 and 20040236268; each of which areherein incorporated by reference in their entireties.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required.

Additionally, the present invention contemplates the use of transdermalpatches, which often have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms may be prepared,for example, by dissolving and/or dispensing the compound in the propermedium. Alternatively or additionally, rate may be controlled by eitherproviding a rate controlling membrane and/or by dispersing the compoundin a polymer matrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.

Topically-administrable formulations may, for example, comprise fromabout 0.1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

Depot Administration

As described herein, in some embodiments, the composition is formulatedin depots for extended release. Generally, a specific organ or tissue (a“target tissue”) is targeted for administration.

In some aspects of the invention, the polynucleotides, primaryconstructs or mmRNA are spatially retained within or proximal to atarget tissue. Provided are method of providing a composition to atarget tissue of a mammalian subject by contacting the target tissue(which contains one or more target cells) with the composition underconditions such that the composition, in particular the nucleic acidcomponent(s) of the composition, is substantially retained in the targettissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90,95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of thecomposition is retained in the target tissue. Advantageously, retentionis determined by measuring the amount of the nucleic acid present in thecomposition that enters one or more target cells. For example, at least1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9,99.99 or greater than 99.99% of the nucleic acids administered to thesubject are present intracellularly at a period of time followingadministration. For example, intramuscular injection to a mammaliansubject is performed using an aqueous composition containing aribonucleic acid and a transfection reagent, and retention of thecomposition is determined by measuring the amount of the ribonucleicacid present in the muscle cells.

Aspects of the invention are directed to methods of providing acomposition to a target tissue of a mammalian subject, by contacting thetarget tissue (containing one or more target cells) with the compositionunder conditions such that the composition is substantially retained inthe target tissue. The composition contains an effective amount of apolynucleotides, primary constructs or mmRNA such that the polypeptideof interest is produced in at least one target cell. The compositionsgenerally contain a cell penetration agent, although “naked” nucleicacid (such as nucleic acids without a cell penetration agent or otheragent) is also contemplated, and a pharmaceutically acceptable carrier.

In some circumstances, the amount of a protein produced by cells in atissue is desirably increased. Preferably, this increase in proteinproduction is spatially restricted to cells within the target tissue.Thus, provided are methods of increasing production of a protein ofinterest in a tissue of a mammalian subject. A composition is providedthat contains polynucleotides, primary constructs or mmRNA characterizedin that a unit quantity of composition has been determined to producethe polypeptide of interest in a substantial percentage of cellscontained within a predetermined volume of the target tissue.

In some embodiments, the composition includes a plurality of differentpolynucleotides, primary constructs or mmRNA, where one or more than oneof the polynucleotides, primary constructs or mmRNA encodes apolypeptide of interest. Optionally, the composition also contains acell penetration agent to assist in the intracellular delivery of thecomposition. A determination is made of the dose of the compositionrequired to produce the polypeptide of interest in a substantialpercentage of cells contained within the predetermined volume of thetarget tissue (generally, without inducing significant production of thepolypeptide of interest in tissue adjacent to the predetermined volume,or distally to the target tissue). Subsequent to this determination, thedetermined dose is introduced directly into the tissue of the mammaliansubject.

In one embodiment, the invention provides for the polynucleotides,primary constructs or mmRNA to be delivered in more than one injectionor by split dose injections.

In one embodiment, the invention may be retained near target tissueusing a small disposable drug reservoir, patch pump or osmotic pump.Non-limiting examples of patch pumps include those manufactured and/orsold by BD® (Franklin Lakes, N.J.), Insulet Corporation (Bedford,Mass.), SteadyMed Therapeutics (San Francisco, Calif.), Medtronic(Minneapolis, Minn.) (e.g., MiniMed), UniLife (York, Pa.), Valeritas(Bridgewater, N.J.), and SpringLeaf Therapeutics (Boston, Mass.). Anon-limiting example of an osmotic pump include those manufactured byDURECT® (Cupertino, Calif.) (e.g., DUROS® and ALZET®).

Pulmonary Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 nm toabout 7 nm or from about 1 nm to about 6 nm. Such compositions aresuitably in the form of dry powders for administration using a devicecomprising a dry powder reservoir to which a stream of propellant may bedirected to disperse the powder and/or using a self propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition. Apropellant may further comprise additional ingredients such as a liquidnon-ionic and/or solid anionic surfactant and/or a solid diluent (whichmay have a particle size of the same order as particles comprising theactive ingredient).

As a non-limiting example, the polynucleotides, primary constructsand/or mmRNA described herein may be formulated for pulmonary deliveryby the methods described in U.S. Pat. No. 8,257,685; herein incorporatedby reference in its entirety.

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Intranasal, nasal and buccal Administration

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 μm to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e. by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein.

Ophthalmic Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis invention. A multilayer thin film device may be prepared to containa pharmaceutical composition for delivery to the eye and/or surroundingtissue.

Payload Administration Detectable Agents and Therapeutic Agents

The polynucleotides, primary constructs or mmRNA described herein can beused in a number of different scenarios in which delivery of a substance(the “payload”) to a biological target is desired, for example deliveryof detectable substances for detection of the target, or delivery of atherapeutic agent. Detection methods can include, but are not limitedto, both imaging in vitro and in vivo imaging methods, e.g.,immunohistochemistry, bioluminescence imaging (BLI), Magnetic ResonanceImaging (MRI), positron emission tomography (PET), electron microscopy,X-ray computed tomography, Raman imaging, optical coherence tomography,absorption imaging, thermal imaging, fluorescence reflectance imaging,fluorescence microscopy, fluorescence molecular tomographic imaging,nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging,photoacoustic imaging, lab assays, or in any situation wheretagging/staining/imaging is required.

The polynucleotides, primary constructs or mmRNA can be designed toinclude both a linker and a payload in any useful orientation. Forexample, a linker having two ends is used to attach one end to thepayload and the other end to the nucleobase, such as at the C-7 or C-8positions of the deaza-adenosine or deaza-guanosine or to the N-3 or C-5positions of cytosine or uracil. The polynucleotide of the invention caninclude more than one payload (e.g., a label and a transcriptioninhibitor), as well as a cleavable linker. In one embodiment, themodified nucleotide is a modified 7-deaza-adenosine triphosphate, whereone end of a cleavable linker is attached to the C7 position of7-deaza-adenine, the other end of the linker is attached to an inhibitor(e.g., to the C5 position of the nucleobase on a cytidine), and a label(e.g., Cy5) is attached to the center of the linker (see, e.g., compoundI of A*pCp C5 Parg Capless in FIG. 5 and columns 9 and 10 of U.S. Pat.No. 7,994,304, incorporated herein by reference). Upon incorporation ofthe modified 7-deaza-adenosine triphosphate to an encoding region, theresulting polynucleotide having a cleavable linker attached to a labeland an inhibitor (e.g., a polymerase inhibitor). Upon cleavage of thelinker (e.g., with reductive conditions to reduce a linker having acleavable disulfide moiety), the label and inhibitor are released.Additional linkers and payloads (e.g., therapeutic agents, detectablelabels, and cell penetrating payloads) are described herein.

Scheme 12 below depicts an exemplary modified nucleotide wherein thenucleobase, adenine, is attached to a linker at the C₁₋₇ carbon of7-deaza adenine. In addition, Scheme 12 depicts the modified nucleotidewith the linker and payload, e.g., a detectable agent, incorporated ontothe 3′ end of the mRNA. Disulfide cleavage and 1,2-addition of the thiolgroup onto the propargyl ester releases the detectable agent. Theremaining structure (depicted, for example, as pApC5 Parg in Scheme 12)is the inhibitor. The rationale for the structure of the modifiednucleotides is that the tethered inhibitor sterically interferes withthe ability of the polymerase to incorporate a second base. Thus, it iscritical that the tether be long enough to affect this function and thatthe inhibiter be in a stereochemical orientation that inhibits orprohibits second and follow on nucleotides into the growingpolynucleotide strand.

For example, the polynucleotides, primary constructs or mmRNA describedherein can be used in reprogramming induced pluripotent stem cells (iPScells), which can directly track cells that are transfected compared tototal cells in the cluster. In another example, a drug that may beattached to the polynucleotides, primary constructs or mmRNA via alinker and may be fluorescently labeled can be used to track the drug invivo, e.g. intracellularly. Other examples include, but are not limitedto, the use of a polynucleotides, primary constructs or mmRNA inreversible drug delivery into cells.

The polynucleotides, primary constructs or mmRNA described herein can beused in intracellular targeting of a payload, e.g., detectable ortherapeutic agent, to specific organelle. Exemplary intracellulartargets can include, but are not limited to, the nuclear localizationfor advanced mRNA processing, or a nuclear localization sequence (NLS)linked to the mRNA containing an inhibitor.

In addition, the polynucleotides, primary constructs or mmRNA describedherein can be used to deliver therapeutic agents to cells or tissues,e.g., in living animals. For example, the polynucleotides, primaryconstructs or mmRNA described herein can be used to deliver highly polarchemotherapeutics agents to kill cancer cells. The polynucleotides,primary constructs or mmRNA attached to the therapeutic agent through alinker can facilitate member permeation allowing the therapeutic agentto travel into a cell to reach an intracellular target.

In one example, the linker is attached at the 2′-position of the ribosering and/or at the 3′ and/or 5′ positionof the polynucleotides, primaryconstructs mmRNA (See e.g., International Pub. No. WO2012030683, hereinincorporated by reference in its entirety). The linker may be any linkerdisclosed herein, known in the art and/or disclosed in InternationalPub. No. WO2012030683, herein incorporated by reference in its entirety.

In another example, the polynucleotides, primary constructs or mmRNA canbe attached to the polynucleotides, primary constructs or mmRNA a viralinhibitory peptide (VIP) through a cleavable linker. The cleavablelinker can release the VIP and dye into the cell. In another example,the polynucleotides, primary constructs or mmRNA can be attached throughthe linker to an ADP-ribosylate, which is responsible for the actions ofsome bacterial toxins, such as cholera toxin, diphtheria toxin, andpertussis toxin. These toxin proteins are ADP-ribosyltransferases thatmodify target proteins in human cells. For example, cholera toxinADP-ribosylates G proteins modifies human cells by causing massive fluidsecretion from the lining of the small intestine, which results inlife-threatening diarrhea.

In some embodiments, the payload may be a therapeutic agent such as acytotoxin, radioactive ion, chemotherapeutic, or other therapeuticagent. A cytotoxin or cytotoxic agent includes any agent that may bedetrimental to cells. Examples include, but are not limited to, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, teniposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein inits entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092,5,585,499, and 5,846,545, all of which are incorporated herein byreference), and analogs or homologs thereof. Radioactive ions include,but are not limited to iodine (e.g., iodine 125 or iodine 131),strontium 89, phosphorous, palladium, cesium, iridium, phosphate,cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

In some embodiments, the payload may be a detectable agent, such asvarious organic small molecules, inorganic compounds, nanoparticles,enzymes or enzyme substrates, fluorescent materials, luminescentmaterials (e.g., luminol), bioluminescent materials (e.g., luciferase,luciferin, and aequorin), chemiluminescent materials, radioactivematerials (e.g., ¹⁸F, ⁶⁷Ga, ^(81m)Kr, ⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl,¹²⁵I ³⁵S, ¹⁴C, ³H, or ^(99m)Tc (e.g., as pertechnetate (technetate(VII),TcO₄ ⁻)), and contrast agents (e.g., gold (e.g., gold nanoparticles),gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagneticiron oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONs), andultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates(e.g., Mn-DPDP), barium sulfate, iodinated contrast media (iohexyl),microbubbles, or perfluorocarbons). Such optically-detectable labelsinclude for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and -6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives(e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red), N,N,N′,N′letramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine, andtetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolic acid;terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

In some embodiments, the detectable agent may be a non-detectablepre-cursor that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). Invitro assays in which the enzyme labeled compositions can be usedinclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), immunoprecipitation assays, immunofluorescence, enzymeimmunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.

Combinations

The polynucleotides, primary constructs or mmRNA may be used incombination with one or more other therapeutic, prophylactic,diagnostic, or imaging agents. By “in combination with,” it is notintended to imply that the agents must be administered at the same timeand/or formulated for delivery together, although these methods ofdelivery are within the scope of the present disclosure. Compositionscan be administered concurrently with, prior to, or subsequent to, oneor more other desired therapeutics or medical procedures. In general,each agent will be administered at a dose and/or on a time scheduledetermined for that agent. In some embodiments, the present disclosureencompasses the delivery of pharmaceutical, prophylactic, diagnostic, orimaging compositions in combination with agents that may improve theirbioavailability, reduce and/or modify their metabolism, inhibit theirexcretion, and/or modify their distribution within the body. As anon-limiting example, the nucleic acids or mmRNA may be used incombination with a pharmaceutical agent for the treatment of cancer orto control hyperproliferative cells. In U.S. Pat. No. 7,964,571, hereinincorporated by reference in its entirety, a combination therapy for thetreatment of solid primary or metastasized tumor is described using apharmaceutical composition including a DNA plasmid encoding forinterleukin-12 with a lipopolymer and also administering at least oneanticancer agent or chemotherapeutic. Further, the nucleic acids andmmRNA of the present invention that encodes anti-proliferative moleculesmay be in a pharmaceutical composition with a lipopolymer (see e.g.,U.S. Pub. No. 20110218231, herein incorporated by reference in itsentirety, claiming a pharmaceutical composition comprising a DNA plasmidencoding an anti-proliferative molecule and a lipopolymer) which may beadministered with at least one chemotherapeutic or anticancer agent.

It will further be appreciated that therapeutically, prophylactically,diagnostically, or imaging active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, it is expected that agentsutilized in combination with be utilized at levels that do not exceedthe levels at which they are utilized individually. In some embodiments,the levels utilized in combination will be lower than those utilizedindividually. In one embodiment, the combinations, each or together maybe administered according to the split dosing regimens described herein.

Dosing

The present invention provides methods comprising administering modifiedmRNAs and their encoded proteins or complexes in accordance with theinvention to a subject in need thereof. Nucleic acids, proteins orcomplexes, or pharmaceutical, imaging, diagnostic, or prophylacticcompositions thereof, may be administered to a subject using any amountand any route of administration effective for preventing, treating,diagnosing, or imaging a disease, disorder, and/or condition (e.g., adisease, disorder, and/or condition relating to working memorydeficits). The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like. Compositions inaccordance with the invention are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention may be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In certain embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg toabout 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging effect. The desired dosage may be deliveredthree times a day, two times a day, once a day, every other day, everythird day, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used.

According to the present invention, it has been discovered thatadministration of mmRNA in split-dose regimens produce higher levels ofproteins in mammalian subjects. As used herein, a “split dose” is thedivision of single unit dose or total daily dose into two or more doses,e.g, two or more administrations of the single unit dose. As usedherein, a “single unit dose” is a dose of any therapeutic administed inone dose/at one time/single route/single point of contact, i.e., singleadministration event. As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose. In one embodiment, the mmRNA of the present invention areadministed to a subject in split doses. The mmRNA may be formulated inbuffer only or in a formulation described herein.

Dosage Forms

A pharmaceutical composition described herein can be formulated into adosage form described herein, such as a topical, intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intracardiac, intraperitoneal,subcutaneous).

Liquid Dosage Forms

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art including, but not limited to, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. In certainembodiments for parenteral administration, compositions may be mixedwith solubilizing agents such as CREMOPHOR, alcohols, oils, modifiedoils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Injectable

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known art andmay include suitable dispersing agents, wetting agents, and/orsuspending agents. Sterile injectable preparations may be sterileinjectable solutions, suspensions, and/or emulsions in nontoxicparenterally acceptable diluents and/or solvents, for example, asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed include, but are not limited to, water, Ringer'ssolution, U.S.P., and isotonic sodium chloride solution. Sterile, fixedoils are conventionally employed as a solvent or suspending medium. Forthis purpose any bland fixed oil can be employed including syntheticmono- or diglycerides. Fatty acids such as oleic acid can be used in thepreparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it may bedesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the polynucleotide,primary construct or mmRNA then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administeredpolynucleotide, primary construct or mmRNA may be accomplished bydissolving or suspending the polynucleotide, primary construct or mmRNAin an oil vehicle. Injectable depot forms are made by formingmicroencapsule matrices of the polynucleotide, primary construct ormmRNA in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of polynucleotide, primary construct or mmRNAto polymer and the nature of the particular polymer employed, the rateof polynucleotide, primary construct or mmRNA release can be controlled.Examples of other biodegradable polymers include, but are not limitedto, poly(orthoesters) and poly(anhydrides). Depot injectableformulations may be prepared by entrapping the polynucleotide, primaryconstruct or mmRNA in liposomes or microemulsions which are compatiblewith body tissues.

Pulmonary

Formulations described herein as being useful for pulmonary delivery mayalso be used for intranasal delivery of a pharmaceutical composition.Another formulation suitable for intranasal administration may be acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 μm to 500 μm. Such a formulation may beadministered in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, contain about 0.1% to 20% (w/w) active ingredient, where thebalance may comprise an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of any additional ingredientsdescribed herein.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21s^(t) ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety).

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. Solid compositions of a similar type may beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like.

Properties of Pharmaceutical Compositions

The pharmaceutical compositions described herein can be characterized byone or more of bioavailability, therapeutic window and/or volume ofdistribution.

Bioavailability

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit anincrease in bioavailability as compared to a composition lacking adelivery agent as described herein. As used herein, the term“bioavailability” refers to the systemic availability of a given amountof polynucleotides, primary constructs or mmRNA administered to amammal. Bioavailability can be assessed by measuring the area under thecurve (AUC) or the maximum serum or plasma concentration (C_(max)) ofthe unchanged form of a compound following administration of thecompound to a mammal. AUC is a determination of the area under the curveplotting the serum or plasma concentration of a compound along theordinate (Y-axis) against time along the abscissa (X-axis). Generally,the AUC for a particular compound can be calculated using methods knownto those of ordinary skill in the art and as described in G. S. Banker,Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72,Marcel Dekker, New York, Inc., 1996, herein incorporated by reference inits entirety.

The C_(max) value is the maximum concentration of the compound achievedin the serum or plasma of a mammal following administration of thecompound to the mammal. The C_(max) value of a particular compound canbe measured using methods known to those of ordinary skill in the art.The phrases “increasing bioavailability” or “improving thepharmacokinetics,” as used herein mean that the systemic availability ofa first polynucleotide, primary construct or mmRNA, measured as AUC,C_(max), or C_(min) in a mammal is greater, when co-administered with adelivery agent as described herein, than when such co-administrationdoes not take place. In some embodiments, the bioavailability of thepolynucleotide, primary construct or mmRNA can increase by at leastabout 2%, at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%.

Therapeutic Window

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit anincrease in the therapeutic window of the administered polynucleotide,primary construct or mmRNA composition as compared to the therapeuticwindow of the administered polynucleotide, primary construct or mmRNAcomposition lacking a delivery agent as described herein. As used herein“therapeutic window” refers to the range of plasma concentrations, orthe range of levels of therapeutically active substance at the site ofaction, with a high probability of eliciting a therapeutic effect. Insome embodiments, the therapeutic window of the polynucleotide, primaryconstruct or mmRNA when co-administered with a delivery agent asdescribed herein can increase by at least about 2%, at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100%.

Volume of Distribution

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit animproved volume of distribution (V_(dist)), e.g., reduced or targeted,relative to a composition lacking a delivery agent as described herein.The volume of distribution (Vdist) relates the amount of the drug in thebody to the concentration of the drug in the blood or plasma. As usedherein, the term “volume of distribution” refers to the fluid volumethat would be required to contain the total amount of the drug in thebody at the same concentration as in the blood or plasma: Vdist equalsthe amount of drug in the body/concentration of drug in blood or plasma.For example, for a 10 mg dose and a plasma concentration of 10 mg/L, thevolume of distribution would be 1 liter. The volume of distributionreflects the extent to which the drug is present in the extravasculartissue. A large volume of distribution reflects the tendency of acompound to bind to the tissue components compared with plasma proteinbinding. In a clinical setting, Vdist can be used to determine a loadingdose to achieve a steady state concentration. In some embodiments, thevolume of distribution of the polynucleotide, primary construct or mmRNAwhen co-administered with a delivery agent as described herein candecrease at least about 2%, at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%.

Biological Effect

In one embodiment, the biological effect of the modified mRNA deliveredto the animals may be categorized by analyzing the protein expression inthe animals. The protein expression may be determined from analyzing abiological sample collected from a mammal administered the modified mRNAof the present invention. In one embodiment, the expression proteinencoded by the modified mRNA administered to the mammal of at least 50pg/ml may be preferred. For example, a protein expression of 50-200pg/ml for the protein encoded by the modified mRNA delivered to themammal may be seen as a therapeutically effective amount of protein inthe mammal.

Detection of Modified Nucleic Acids by Mass Spectrometry

Mass spectrometry (MS) is an analytical technique that can providestructural and molecular mass/concentration information on moleculesafter their conversion to ions. The molecules are first ionized toacquire positive or negative charges and then they travel through themass analyzer to arrive at different areas of the detector according totheir mass/charge (m/z) ratio.

Mass spectrometry is performed using a mass spectrometer which includesan ion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. For example ionization of the sample maybe performed by electrospray ionization (ESI), atmospheric pressurechemical ionization (APCI), photoionization, electron ionization, fastatom bombardment (FAB)/liquid secondary ionization (LSIMS), matrixassisted laser desorption/ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, and particle beamionization. The skilled artisan will understand that the choice ofionization method can be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z). Suitable analyzers for determiningmass-to-charge ratios include quadropole analyzers, ion traps analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected (i.e., usinga selective ion monitoring mode (SIM)), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM).

Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupledwith stable isotope labeled dilution of peptide standards has been shownto be an effective method for protein verification (e.g., Keshishian etal., Mol Cell Proteomics 2009 8: 2339-2349; Kuhn et al., Clin Chem 200955:1108-1117; Lopez et al., Clin Chem 2010 56:281-290; each of which areherein incorporated by reference in its entirety). Unlike untargetedmass spectrometry frequently used in biomarker discovery studies,targeted MS methods are peptide sequence-based modes of MS that focusthe full analytical capacity of the instrument on tens to hundreds ofselected peptides in a complex mixture. By restricting detection andfragmentation to only those peptides derived from proteins of interest,sensitivity and reproducibility are improved dramatically compared todiscovery-mode MS methods. This method of mass spectrometry-basedmultiple reaction monitoring (MRM) quantitation of proteins candramatically impact the discovery and quantitation of biomarkers viarapid, targeted, multiplexed protein expression profiling of clinicalsamples.

In one embodiment, a biological sample which may contain at least oneprotein encoded by at least one modified mRNA of the present inventionmay be analyzed by the method of MRM-MS. The quantification of thebiological sample may further include, but is not limited to,isotopically labeled peptides or proteins as internal standards.

According to the present invention, the biological sample, once obtainedfrom the subject, may be subjected to enzyme digestion. As used herein,the term “digest” means to break apart into shorter peptides. As usedherein, the phrase “treating a sample to digest proteins” meansmanipulating a sample in such a way as to break down proteins in asample. These enzymes include, but are not limited to, trypsin,endoproteinase Glu-C and chymotrypsin. In one embodiment, a biologicalsample which may contain at least one protein encoded by at least onemodified mRNA of the present invention may be digested using enzymes.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for proteinusing electrospray ionization. Electrospray ionization (ESI) massspectrometry (ESIMS) uses electrical energy to aid in the transfer ofions from the solution to the gaseous phase before they are analyzed bymass spectrometry. Samples may be analyzed using methods known in theart (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12; hereinincorporated by reference in its entirety). The ionic species containedin solution may be transferred into the gas phase by dispersing a finespray of charge droplets, evaporating the solvent and ejecting the ionsfrom the charged droplets to generate a mist of highly charged droplets.The mist of highly charged droplets may be analyzed using at least 1, atleast 2, at least 3 or at least 4 mass analyzers such as, but notlimited to, a quadropole mass analyzer. Further, the mass spectrometrymethod may include a purification step. As a non-limiting example, thefirst quadrapole may be set to select a single m/z ratio so it mayfilter out other molecular ions having a different m/z ratio which mayeliminate complicated and time-consuming sample purification proceduresprior to MS analysis.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for protein ina tandem ESIMS system (e.g., MS/MS). As non-limiting examples, thedroplets may be analyzed using a product scan (or daughter scan) aprecursor scan (parent scan) a neutral loss or a multiple reactionmonitoring.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed usingmatrix-assisted laser desorption/ionization (MALDI) mass spectrometry(MALDIMS). MALDI provides for the nondestructive vaporization andionization of both large and small molecules, such as proteins. In MALDIanalysis, the analyte is first co-crystallized with a large molar excessof a matrix compound, which may also include, but is not limited to, anultraviolet absorbing weak organic acid. Non-limiting examples ofmatrices used in MALDI are α-cyano-4-hydroxycinnamic acid,3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.Laser radiation of the analyte-matrix mixture may result in thevaporization of the matrix and the analyte. The laser induced desorptionprovides high ion yields of the intact analyte and allows formeasurement of compounds with high accuracy. Samples may be analyzedusing methods known in the art (e.g., Lewis, Wei and Siuzdak,Encyclopedia of Analytical Chemistry 2000:5880-5894; herein incorporatedby reference in its entirety). As non-limiting examples, mass analyzersused in the MALDI analysis may include a linear time-of-flight (TOF), aTOF reflectron or a Fourier transform mass analyzer.

In one embodiment, the analyte-matrix mixture may be formed using thedried-droplet method. A biologic sample is mixed with a matrix to createa saturated matrix solution where the matrix-to-sample ratio isapproximately 5000:1. An aliquot (approximately 0.5-2.0 uL) of thesaturated matrix solution is then allowed to dry to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethin-layer method. A matrix homogeneous film is first formed and thenthe sample is then applied and may be absorbed by the matrix to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethick-layer method. A matrix homogeneous film is formed with anitro-cellulose matrix additive. Once the uniform nitro-cellulose matrixlayer is obtained the sample is applied and absorbed into the matrix toform the analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thesandwich method. A thin layer of matrix crystals is prepared as in thethin-layer method followed by the addition of droplets of aqueoustrifluoroacetic acid, the sample and matrix. The sample is then absorbedinto the matrix to form the analyte-matrix mixture.

V. Uses of Polynucleotides, primary constructs and mmRNA of theInvention

The polynucleotides, primary constructs and mmRNA of the presentinvention are designed, in preferred embodiments, to provide foravoidance or evasion of deleterious bio-responses such as the immuneresponse and/or degradation pathways, overcoming the threshold ofexpression and/or improving protein production capacity, improvedexpression rates or translation efficiency, improved drug or proteinhalf life and/or protein concentrations, optimized protein localization,to improve one or more of the stability and/or clearance in tissues,receptor uptake and/or kinetics, cellular access by the compositions,engagement with translational machinery, secretion efficiency (whenapplicable), accessibility to circulation, and/or modulation of a cell'sstatus, function and/or activity.

Therapeutics Therapeutic Agents

The polynucleotides, primary constructs or mmRNA of the presentinvention, such as modified nucleic acids and modified RNAs, and theproteins translated from them described herein can be used astherapeutic or prophylactic agents. They are provided for use inmedicine. For example, a polynucleotide, primary construct or mmRNAdescribed herein can be administered to a subject, wherein thepolynucleotide, primary construct or mmRNA is translated in vivo toproduce a therapeutic or prophylactic polypeptide in the subject.Provided are compositions, methods, kits, and reagents for diagnosis,treatment or prevention of a disease or condition in humans and othermammals. The active therapeutic agents of the invention includepolynucleotides, primary constructs or mmRNA, cells containingpolynucleotides, primary constructs or mmRNA or polypeptides translatedfrom the polynucleotides, primary constructs or mmRNA.

In certain embodiments, provided herein are combination therapeuticscontaining one or more polynucleotide, primary construct or mmRNAcontaining translatable regions that encode for a protein or proteinsthat boost a mammalian subject's immunity along with a protein thatinduces antibody-dependent cellular toxicity. For example, providedherein are therapeutics containing one or more nucleic acids that encodetrastuzumab and granulocyte-colony stimulating factor (G-CSF). Inparticular, such combination therapeutics are useful in Her2+ breastcancer patients who develop induced resistance to trastuzumab. (See,e.g., Albrecht, Immunotherapy. 2(6):795-8 (2010)).

Provided herein are methods of inducing translation of a recombinantpolypeptide in a cell population using the polynucleotide, primaryconstruct or mmRNA described herein. Such translation can be in vivo, exvivo, in culture, or in vitro. The cell population is contacted with aneffective amount of a composition containing a nucleic acid that has atleast one nucleoside modification, and a translatable region encodingthe recombinant polypeptide. The population is contacted underconditions such that the nucleic acid is localized into one or morecells of the cell population and the recombinant polypeptide istranslated in the cell from the nucleic acid.

An “effective amount” of the composition is provided based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the nucleic acid (e.g., size, and extent ofmodified nucleosides), and other determinants. In general, an effectiveamount of the composition provides efficient protein production in thecell, preferably more efficient than a composition containing acorresponding unmodified nucleic acid. Increased efficiency may bedemonstrated by increased cell transfection (i.e., the percentage ofcells transfected with the nucleic acid), increased protein translationfrom the nucleic acid, decreased nucleic acid degradation (asdemonstrated, e.g., by increased duration of protein translation from amodified nucleic acid), or reduced innate immune response of the hostcell.

Aspects of the invention are directed to methods of inducing in vivotranslation of a recombinant polypeptide in a mammalian subject in needthereof. Therein, an effective amount of a composition containing anucleic acid that has at least one structural or chemical modificationand a translatable region encoding the recombinant polypeptide isadministered to the subject using the delivery methods described herein.The nucleic acid is provided in an amount and under other conditionssuch that the nucleic acid is localized into a cell of the subject andthe recombinant polypeptide is translated in the cell from the nucleicacid. The cell in which the nucleic acid is localized, or the tissue inwhich the cell is present, may be targeted with one or more than onerounds of nucleic acid administration.

In certain embodiments, the administered polynucleotide, primaryconstruct or mmRNA directs production of one or more recombinantpolypeptides that provide a functional activity which is substantiallyabsent in the cell, tissue or organism in which the recombinantpolypeptide is translated. For example, the missing functional activitymay be enzymatic, structural, or gene regulatory in nature. In relatedembodiments, the administered polynucleotide, primary construct or mmRNAdirects production of one or more recombinant polypeptides thatincreases (e.g., synergistically) a functional activity which is presentbut substantially deficient in the cell in which the recombinantpolypeptide is translated.

In other embodiments, the administered polynucleotide, primary constructor mmRNA directs production of one or more recombinant polypeptides thatreplace a polypeptide (or multiple polypeptides) that is substantiallyabsent in the cell in which the recombinant polypeptide is translated.Such absence may be due to genetic mutation of the encoding gene orregulatory pathway thereof. In some embodiments, the recombinantpolypeptide increases the level of an endogenous protein in the cell toa desirable level; such an increase may bring the level of theendogenous protein from a subnormal level to a normal level or from anormal level to a super-normal level.

Alternatively, the recombinant polypeptide functions to antagonize theactivity of an endogenous protein present in, on the surface of, orsecreted from the cell. Usually, the activity of the endogenous proteinis deleterious to the subject; for example, due to mutation of theendogenous protein resulting in altered activity or localization.Additionally, the recombinant polypeptide antagonizes, directly orindirectly, the activity of a biological moiety present in, on thesurface of, or secreted from the cell. Examples of antagonizedbiological moieties include lipids (e.g., cholesterol), a lipoprotein(e.g., low density lipoprotein), a nucleic acid, a carbohydrate, aprotein toxin such as shiga and tetanus toxins, or a small moleculetoxin such as botulinum, cholera, and diphtheria toxins. Additionally,the antagonized biological molecule may be an endogenous protein thatexhibits an undesirable activity, such as a cytotoxic or cytostaticactivity.

The recombinant proteins described herein may be engineered forlocalization within the cell, potentially within a specific compartmentsuch as the nucleus, or are engineered for secretion from the cell ortranslocation to the plasma membrane of the cell.

In some embodiments, modified mRNAs and their encoded polypeptides inaccordance with the present invention may be used for treatment of anyof a variety of diseases, disorders, and/or conditions, including butnot limited to one or more of the following: autoimmune disorders (e.g.diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis);inflammatory disorders (e.g. arthritis, pelvic inflammatory disease);infectious diseases (e.g. viral infections (e.g., HIV, HCV, RSV),bacterial infections, fungal infections, sepsis); neurological disorders(e.g. Alzheimer's disease, Huntington's disease; autism; Duchennemuscular dystrophy); cardiovascular disorders (e.g. atherosclerosis,hypercholesterolemia, thrombosis, clotting disorders, angiogenicdisorders such as macular degeneration); proliferative disorders (e.g.cancer, benign neoplasms); respiratory disorders (e.g. chronicobstructive pulmonary disease); digestive disorders (e.g. inflammatorybowel disease, ulcers); musculoskeletal disorders (e.g. fibromyalgia,arthritis); endocrine, metabolic, and nutritional disorders (e.g.diabetes, osteoporosis); urological disorders (e.g. renal disease);psychological disorders (e.g. depression, schizophrenia); skin disorders(e.g. wounds, eczema); blood and lymphatic disorders (e.g. anemia,hemophilia); etc.

Diseases characterized by dysfunctional or aberrant protein activityinclude cystic fibrosis, sickle cell anemia, epidermolysis bullosa,amyotrophic lateral sclerosis, and glucose-6-phosphate dehydrogenasedeficiency. The present invention provides a method for treating suchconditions or diseases in a subject by introducing nucleic acid orcell-based therapeutics containing the polynucleotide, primary constructor mmRNA provided herein, wherein the polynucleotide, primary constructor mmRNA encode for a protein that antagonizes or otherwise overcomesthe aberrant protein activity present in the cell of the subject.Specific examples of a dysfunctional protein are the missense mutationvariants of the cystic fibrosis transmembrane conductance regulator(CFTR) gene, which produce a dysfunctional protein variant of CFTRprotein, which causes cystic fibrosis.

Diseases characterized by missing (or substantially diminished such thatproper (normal or physiological protein function does not occur) proteinactivity include cystic fibrosis, Niemann-Pick type C, β thalassemiamajor, Duchenne muscular dystrophy, Hurler Syndrome, Hunter Syndrome,and Hemophilia A. Such proteins may not be present, or are essentiallynon-functional. The present invention provides a method for treatingsuch conditions or diseases in a subject by introducing nucleic acid orcell-based therapeutics containing the polynucleotide, primary constructor mmRNA provided herein, wherein the polynucleotide, primary constructor mmRNA encode for a protein that replaces the protein activity missingfrom the target cells of the subject. Specific examples of adysfunctional protein are the nonsense mutation variants of the cysticfibrosis transmembrane conductance regulator (CFTR) gene, which producea nonfunctional protein variant of CFTR protein, which causes cysticfibrosis.

Thus, provided are methods of treating cystic fibrosis in a mammaliansubject by contacting a cell of the subject with a polynucleotide,primary construct or mmRNA having a translatable region that encodes afunctional CFTR polypeptide, under conditions such that an effectiveamount of the CTFR polypeptide is present in the cell. Preferred targetcells are epithelial, endothelial and mesothelial cells, such as thelung, and methods of administration are determined in view of the targettissue; i.e., for lung delivery, the RNA molecules are formulated foradministration by inhalation.

In another embodiment, the present invention provides a method fortreating hyperlipidemia in a subject, by introducing into a cellpopulation of the subject with a modified mRNA molecule encodingSortilin, a protein recently characterized by genomic studies, therebyameliorating the hyperlipidemia in a subject. The SORT1 gene encodes atrans-Golgi network (TGN) transmembrane protein called Sortilin. Geneticstudies have shown that one of five individuals has a single nucleotidepolymorphism, rs12740374, in the 1p13 locus of the SORT1 gene thatpredisposes them to having low levels of low-density lipoprotein (LDL)and very-low-density lipoprotein (VLDL). Each copy of the minor allele,present in about 30% of people, alters LDL cholesterol by 8 mg/dL, whiletwo copies of the minor allele, present in about 5% of the population,lowers LDL cholesterol 16 mg/dL. Carriers of the minor allele have alsobeen shown to have a 40% decreased risk of myocardial infarction.Functional in vivo studies in mice describes that overexpression ofSORT1 in mouse liver tissue led to significantly lower LDL-cholesterollevels, as much as 80% lower, and that silencing SORT1 increased LDLcholesterol approximately 200% (Musunuru K et al. From noncoding variantto phenotype via SORT1 at the 1p13 cholesterol locus. Nature 2010; 466:714-721).

In another embodiment, the present invention provides a method fortreating hematopoietic disorders, cardiovascular disease, oncology,diabetes, cystic fibrosis, neurological diseases, inborn errors ofmetabolism, skin and systemic disorders, and blindness. The identity ofmolecular targets to treat these specific diseases has been described(Templeton ed., Gene and Cell Therapy: Therapeutic Mechanisms andStrategies, 3^(rd) Edition, Bota Raton, Fla.:CRC Press; hereinincorporated by reference in its entirety).

Provided herein, are methods to prevent infection and/or sepsis in asubject at risk of developing infection and/or sepsis, the methodcomprising administering to a subject in need of such prevention acomposition comprising a polynucleotide, primary construct or mmRNAprecursor encoding an anti-microbial polypeptide (e.g., ananti-bacterial polypeptide), or a partially or fully processed formthereof in an amount sufficient to prevent infection and/or sepsis. Incertain embodiments, the subject at risk of developing infection and/orsepsis may be a cancer patient. In certain embodiments, the cancerpatient may have undergone a conditioning regimen. In some embodiments,the conditioning regiment may include, but is not limited to,chemotherapy, radiation therapy, or both. As a non-limiting example, apolynucleotide, primary construct or mmRNA can encode Protein C, itszymogen or prepro-protein, the activated form of Protein C (APC) orvariants of Protein C which are known in the art. The polynucleotides,primary constructs or mmRNA may be chemically modified and delivered tocells. Non-limiting examples of polypeptides which may be encoded withinthe chemically modified mRNAs of the present invention include thosetaught in U.S. Pat. Nos. 7,226,999; 7,498,305; 6,630,138 each of whichis incorporated herein by reference in its entirety. These patents teachProtein C like molecules, variants and derivatives, any of which may beencoded within the chemically modified molecules of the presentinvention.

Further provided herein, are methods to treat infection and/or sepsis ina subject, the method comprising administering to a subject in need ofsuch treatment a composition comprising a polynucleotide, primaryconstruct or mmRNA precursor encoding an anti-microbial polypeptide(e.g., an anti-bacterial polypeptide), e.g., an anti-microbialpolypeptide described herein, or a partially or fully processed formthereof in an amount sufficient to treat an infection and/or sepsis. Incertain embodiments, the subject in need of treatment is a cancerpatient. In certain embodiments, the cancer patient has undergone aconditioning regimen. In some embodiments, the conditioning regiment mayinclude, but is not limited to, chemotherapy, radiation therapy, orboth.

In certain embodiments, the subject may exhibits acute or chronicmicrobial infections (e.g., bacterial infections). In certainembodiments, the subject may have received or may be receiving atherapy. In certain embodiments, the therapy may include, but is notlimited to, radiotherapy, chemotherapy, steroids, ultraviolet radiation,or a combination thereof. In certain embodiments, the patient may sufferfrom a microvascular disorder. In some embodiments, the microvasculardisorder may be diabetes. In certain embodiments, the patient may have awound. In some embodiments, the wound may be an ulcer. In a specificembodiment, the wound may be a diabetic foot ulcer. In certainembodiments, the subject may have one or more burn wounds. In certainembodiments, the administration may be local or systemic. In certainembodiments, the administration may be subcutaneous. In certainembodiments, the administration may be intravenous. In certainembodiments, the administration may be oral. In certain embodiments, theadministration may be topical. In certain embodiments, theadministration may be by inhalation. In certain embodiments, theadministration may be rectal. In certain embodiments, the administrationmay be vaginal.

Other aspects of the present disclosure relate to transplantation ofcells containing polynucleotide, primary construct, or mmRNA to amammalian subject. Administration of cells to mammalian subjects isknown to those of ordinary skill in the art, and include, but is notlimited to, local implantation (e.g., topical or subcutaneousadministration), organ delivery or systemic injection (e.g., intravenousinjection or inhalation), and the formulation of cells inpharmaceutically acceptable carrier. Such compositions containingpolynucleotide, primary construct, or mmRNA can be formulated foradministration intramuscularly, transarterially, intraperitoneally,intravenously, intranasally, subcutaneously, endoscopically,transdermally, or intrathecally. In some embodiments, the compositionmay be formulated for extended release.

The subject to whom the therapeutic agent may be administered suffersfrom or may be at risk of developing a disease, disorder, or deleteriouscondition. Provided are methods of identifying, diagnosing, andclassifying subjects on these bases, which may include clinicaldiagnosis, biomarker levels, genome-wide association studies (GWAS), andother methods known in the art.

Rare Liver Diseases or Disorders Progressive Familial IntrahepaticCholestasis (PFIC)

In one embodiment, the rare liver disease or disorder polynucleotide,primary construct, or mmRNA of the present invention may be used totreat progressive familial intrahepatic cholestasis (PFIC). The term“progressive familial intrahepatic cholestasis” or “PFIC” as used hereinrefers to a liver disorder that can lead to liver failure. PFIC ischaracterized by autosomal recessive defects in bile formation andhepatocellular cholestasis. As used herein, “hepatocellular” is a termused to describe something that affects or pertains to liver cells (alsoreferred to as hepatocytes). As used herein, the term “cholestasis”refers to a condition characterized by slowed or disrupted flow of bilefrom the liver. As used herein, the term “bile” refers to a liquidsubstance produced by the liver comprising water, bile salts, mucus,fats inorganic salts and cholesterol, which aids in the emulsificationand digestion of dietary fats.

There are three known types of PFIC(PFIC-1, PFIC-2 and PFIC-3), and theyhave been traced to mutations in genes encoding proteins involved in thehepatocellular transport system and in the formation of bile.

PFIC-1 and PFIC-2 are typically diagnosed in infancy but diagnosis mayoccur during the prenatal period or the neonatal period. As used herein,the term “prenatal” refers to the period of time in the life of anorganism that occurs before birth. As used herein, the term “neonatal”refers to a period of time in the life of an organism that occurs afterbirth. In humans, the neonatal period may include a period of time frombirth to about 1 month, to about 3 months or to about 6 months afterbirth. As used herein, the term “infancy” refers to a period in the lifeof an organism that occurs between birth and childhood. In humans,infancy may include the period of time from birth to about 1 year oflife, to about 2 years of life, to about 3 years of life or to about 4years of life.

PFIC-3 may be diagnosed during the prenatal period, during the neonatalperiod or during infancy. In some cases, PFIC-3 may be able evadediagnosis until childhood or young adulthood. As used herein, the term“childhood” refers to the period of time in the life of an organism thatoccurs after infancy and before young adulthood. In humans, childhoodmay include the period of time from about 2 years of age to about 10years of age, from about 3 years of age to about 11 years of age, fromabout 4 years of age to about 12 years of age or from about 5 years ofage to about 13 years of age. As used herein, the term “young adulthood”refers to the period of time in the life of an organism betweenchildhood and adulthood. In humans, young adulthood may include theperiods of time from about 10 years of age to about 16 years of age,from about 11 years of age to about 17 years of age, from about 12 yearsof age to about 18 years of age, from about 13 years of age to about 19years of age and from about 14 years of age to about 20 years of age.

Clinical manifestations of PFIC include, but are not limited topruritus, cholestasis and jaundice. As used herein, the term “pruritus”refers to an uncomfortable sensation that leads to the urge to scratchor rub an afflicted part of the body. As used herein, the term“jaundice” refers to a condition characterized by yellow skin, eyesand/or mucus membranes resulting from the buildup of bilirubin.

Most patients with PFIC develop fibrosis and suffer from liver failureprior to adulthood. Individuals afflicted with PFIC may be diagnosedthrough observation of clinical manifestations, cholangiography, liverultrasonography, liver histology and genetic testing. Additional testsmay be conducted to exclude other disorders resulting in childhoodcholestasis.

Dysfunction in one of three different cellular transporters causes eachof PFIC types 1, 2 and 3. Each is involved in lipid transport and eachplays a vital role in the secretion of bile from the liver. PFIC type 1is caused by genetic mutations that lead to dysfunction of ATPaseaminophospholipid transporter, class I, type 8B, member 1 (ATP8B1).ATP8B1 functions to translocate phosphatidylserine andphosphatidylethanolamine from one side of a phospholipid bilayer to theother. Both PFIC-2 and PFIC-3 are caused by mutations affecting thefunction of ATP-binding cassette (ABC) transporters. ABC, subfamily B,member 11 (ABCB11) transporters support bile production through thetransport of taurocholate as well as other cholate-linked compounds fromhepatocytes into the bile. Defective ABCB11 function leads to PFIC-2.ABC subfamily B, member 4 (ABCB4) transports phospholipids, preferablyphosphatidyl choline, across hepatocellular membranes for incorporationinto bile. Gene mutations that lead to dysfunction of ABCB4 areresponsible for PFIC-3. (Davit-Spraul, A. et al., Progressive familialintrahepatic cholestasis. Orphanet J Rare Dis. 2009 Jan. 8; 4:1;Degiorgio, D. et al., Molecular characterization and structuralimplications of 25 new ABCB4 mutations in progressive familialintrahepatic cholestasis type 3 (PFIC3). Eur J Hum Genet. 2007 December;15(12):1230-8; each of which are herein incorporated by reference intheir entireties).

In one embodiment, patients with PFIC may be administered a compositioncomprising at least one rare liver disease or disorder polynucleotide,primary construct or mmRNA of the present invention. The rare liverdisease or disorder polynucleotide, primary construct or mmRNA mayencode a peptide, protein or fragment thereof such as, but not limitedto, ATP-binding cassette, sub-family (MDR/TAP), member 4 (ABCB4),ATP-binding cassette, sub-family (MDR/TAP, member 11 (ABCB11) andATPase, aminophopholipid transporter, class I, type 8B, member 1(ATP8B1).

In one embodiment, PFIC-1 may be treated by administering a compositionof the present invention comprising at least one rare liver disease ordisorder polynucleotide, primary construct or mmRNA encoding a peptide,protein or fragment thereof of ATP8B1. In another embodiment, PFIC-1 maybe treated by administering a composition of the present inventioncomprising at least one rare liver disease or disorder polynucleotide,primary construct or mmRNA encoding a peptide, protein or fragmentthereof comprising SEQ ID NO: 855 or 856.

In one embodiment, PFIC-2 may be treated by administering a compositionof the present invention comprising at least one rare liver disease ordisorder polynucleotide, primary construct or mmRNA encoding a peptide,protein or fragment thereof of ABCB11. In another embodiment, PFIC-2 maybe treated by administering a composition of the present inventioncomprising at least one rare liver disease or disorder polynucleotide,primary construct or mmRNA encoding a peptide, protein or fragmentthereof comprising SEQ ID NO: 865.

In one embodiment, PFIC-3 may be treated by administering a compositionof the present invention comprising at least one rare liver disease ordisorder polynucleotide, primary construct or mmRNA encoding a peptide,protein or fragment thereof of ABCB4. In another embodiment, PFIC-3 maybe treated by administering a composition of the present inventioncomprising at least one rare liver disease or disorder polynucleotide,primary construct or mmRNA encoding a peptide, protein or fragmentthereof comprising SEQ ID NO: 866-871.

Familial Hypercholesterolemia

In one embodiment, the rare liver disease or disorder polynucleotide,primary construct, or mmRNA of the present invention may be used totreat familial hypercholesterolemia (FH). As used herein, the term“familial hypercholesterolemia” or “FH” refers to an inherited disordercharacterized by elevated levels of low density lipoprotein(LDL)-associated cholesterol in the plasma. Patients or subjects with FHmay have an increased risk for cardiovascular disease at a young age. Insome embodiments, high levels of LDL in the blood of these individualsmay be the result of mutations in the gene encoding the LDL receptor. Itis believed, and is no means limiting, that the LDL receptor binds LDLin the circulation and facilitates endocytosis of LDL into the cell thatthe receptor is expressed on. When this receptor is dysfunctional, LDLlevels remain elevated in the circulation and promote the development ofatherosclerosis. Individuals with FH may be heterozygous or homozygousfor FH-related gene mutations. Symptoms in homozygous individuals can bemore severe. Diagnosis is possible during childhood or young adulthoodby methods known in the art including, but not limited to, a physicalexam that reveals xanthomas (fatty skin growths). Earlier diagnosis ofFH may be made through an analysis of family history and genetics.(Sjouke, B. et al., Familial hypercholesterolemia: present and futuremanagement. Curr Cardiol Rep. 2011 December; 13(6):527-36; Avis, H. J.et al., A systematic review and meta-analysis of statin therapy inchildren with familial hypercholesterolemia. Arterioscler Thromb VascBiol. 2007 August; 27(8):1803-10; each of which are herein incorporatedby reference in their entireties).

In one embodiment, patients with FH may be administered a compositioncomprising at least one rare liver disease or disorder polynucleotide,primary construct or mmRNA of the present invention. The rare liverdisease or disorder polynucleotide, primary construct or mmRNA mayencode a peptide, protein or fragment thereof such as, but not limitedto, low density lipoprotein receptor (LDLR), apolipoprotein B (APOB),and proprotein convertase subtilisin/kexin type 9 (PCSK9).

In one embodiment, FH may be treated by administering a composition ofthe present invention comprising at least one rare liver disease ordisorder polynucleotide, primary construct or mmRNA encoding a peptide,protein or fragment thereof of LDLR. In another embodiment, FH may betreated by administering a composition of the present inventioncomprising at least one rare liver disease or disorder polynucleotide,primary construct or mmRNA encoding a peptide, protein or fragmentthereof comprising SEQ ID NO: 1145-1151.

In one embodiment, FH may be treated by administering a composition ofthe present invention comprising at least one rare liver disease ordisorder polynucleotide, primary construct or mmRNA encoding a peptide,protein or fragment thereof of APOB. In another embodiment, FH may betreated by administering a composition of the present inventioncomprising at least one rare liver disease or disorder polynucleotide,primary construct or mmRNA encoding a peptide, protein or fragmentthereof comprising SEQ ID NO: 819 or 819.

In one embodiment, FH may be treated by administering a composition ofthe present invention comprising at least one rare liver disease ordisorder polynucleotide, primary construct or mmRNA encoding a peptide,protein or fragment thereof of PCSK9. In another embodiment, FH may betreated by administering a composition of the present inventioncomprising at least one rare liver disease or disorder polynucleotide,primary construct or mmRNA encoding a peptide, protein or fragmentthereof comprising SEQ ID NO: 1241-1243.

Ornithine transcarbamylase Deficiency

In one embodiment, the rare liver disease or disorder polynucleotide,primary construct, or mmRNA of the present invention may be used totreat ornithine transcarbamylase deficiency (OTCD). As used herein, theterm “ornithine transcarbamylase deficiency” or “OTCD” refers to aninherited disorder in the synthesis of urea resulting from mutations inthe gene encoding the enzyme ornithine transcarbamylase (OTC). Thesemutations can lead to hyperammonemia, neurological problems and anincreased rate of death. OTC is an important component of the ureacycle, catalyzing the formation of citrulline from carbamoyl phosphateand ornithine. This process is critical to the removal of excess ammoniafrom the body. The gene for OTC is carried on the X chromosome makingOTCD an X-linked disorder. As with most X-linked disorders, OTCDprimarily affects males, while females are carriers. The severity ofOTCD depends upon the nature of the mutation in the gene. Gene mutationsin individuals that result in non-functional OTC typically lead to thedeath of those individuals within the first month of life. Suchmutations may be discovered by genetic analysis in an individualsuspected of suffering from OTCD. Individuals with gene mutations thatlead to varying degrees of enzyme function may survive longer andrespond to treatments to reduce the effects of the disease. Diagnosis ofOTCD may occur after the observation of symptoms and the detection ofhigh levels of ammonia and orotic acid in the urine. (Brunetti-Pierri,N. et al., Phenotypic correction of ornithine transcarbamylasedeficiency using low dose helper-dependent adenoviral vectors. J GeneMed. 2008 August; 10(8):890-6; Wilmslow, U.K., Ornithinetranscarbamylase deficiency: a urea cycle defect. Eur J Paediatr Neurol.2003; 7(3):115-21; each of which are herein incorporated by reference intheir entireties).

In one embodiment, patients with OTC may be administered a compositioncomprising at least one rare liver disease or disorder polynucleotide,primary construct or mmRNA of the present invention. The rare liverdisease or disorder polynucleotide, primary construct or mmRNA mayencode a peptide, protein or fragment thereof such as, but not limitedto, ornithine carbamoyltransferase (OTC).

In one embodiment, OTCD may be treated by administering a composition ofthe present invention comprising at least one rare liver disease ordisorder polynucleotide, primary construct or mmRNA encoding a peptide,protein or fragment thereof of OTC.

In another embodiment, OTCD may be treated by administering acomposition of the present invention comprising at least one rare liverdisease or disorder polynucleotide, primary construct or mmRNA encodinga peptide, protein or fragment thereof comprising SEQ ID NO: 1192.

Crigler-Najjar Syndrome

In one embodiment, the rare liver disease or disorder polynucleotide,primary construct, or mmRNA of the present invention may be used totreat Crigler-Najjar syndrome. As used herein, the term “Crigler-Najjarsyndrome” refers to an inborn error affecting the function of the 1A1isoform of the enzyme bilirubin-uridine diphosphateglucuronosyltransferase (UGT1A1). UGT1A1 is critical in thedetoxification of hydrophobic bilirubin, a toxic byproduct of hemedegradation. It is believed that UGT1A1 functions by linking hydrophobicbilirubin to glucoronic acid for excretion into the bile. Individualssuffering from Crigler-Najjar syndrome can experience unconjugatedhyperbilirubinemia (or excess hydrophobic bilirubin in the circulation),leading to neurological impairment and the requirement for regularphototherapy that becomes less and less effective as the patients age.Diagnosis of Crigler-Najjar syndrome typically begins with theobservation of jaundice in infants and then enzyme function may beevaluated by enzyme and liver assays known in the art. (Lysy, P. A. etal., Liver cell transplantation for Crigler-Najjar syndrome type I:update and perspectives. World J Gastroenterol. 2008 Jun. 14;14(22):3464-70; Sugatani, J., Function, genetic polymorphism, andtranscriptional regulation of human UDP-glucuronosyltransferase (UGT)1A1. Drug Metab Pharmacokinet 2012 Oct. 23. [Epub ahead of print]; eachof which are herein incorporated by reference in their entireties).

In one embodiment, patients with Crigler-Najjar syndrome may beadministered a composition comprising at least one rare liver disease ordisorder polynucleotide, primary construct or mmRNA of the presentinvention. The rare liver disease or disorder polynucleotide, primaryconstruct or mmRNA may encode a peptide, protein or fragment thereofsuch as, but not limited to, UGT1A1.

In one embodiment, Crigler-Najjar syndrome may be treated byadministering a composition of the present invention comprising at leastone rare liver disease or disorder polynucleotide, primary construct ormmRNA encoding a peptide, protein or fragment thereof of UGT1A1. Inanother embodiment, Crigler-Najjar syndrome may be treated byadministering a composition of the present invention comprising at leastone rare liver disease or disorder polynucleotide, primary construct ormmRNA encoding a peptide, protein or fragment thereof comprising SEQ IDNO: 1357 or 1358.

Aceruloplasminemia

In one embodiment, the rare liver disease or disorder polynucleotide,primary construct, or mmRNA of the present invention may be used totreat Aceruloplasminemia. As used herein, the term “aceruloplasminemia”refers to a disease in which ceruloplasmin (CP) may be defective and/ordysfunctional due to a gene mutation in the CP gene. It is believed thatCP is a transport protein responsible for transporting iron from cellsinto capillaries. Once in the capillaries, iron can bind to ferritin andenters the blood stream. In the absence of functional CP, ironaccumulates within cells and serum ferritin levels become elevated.(Ogimoto, M. et al., Criteria for early identification ofaceruloplasminemia. Intern Med. 2011; 50(13):1415-8; Miyajima, H.,Aceruloplasminemia. GeneReviews [Internet]. Seattle (WA): University ofWashington, Seattle; 2003 August 12 [updated 2011 Feb. 17]; each ofwhich are herein incorporated by reference in its entirety).

Iron accumulation may be especially severe in the liver as well as inthe eye, brain and pancreas. Aceruloplasminemia, arising from geneticdefects in the CP gene, is an autosomal recessive disorder. As usedherein, the term “autosomal rescessive” refers to a disease, disorder,trait or phenotype that can affect those who are homozygous for aresponsible gene. Symptoms of the disease typically manifest between theages of 25 and 60 years old. Diagnosis of individuals believed to besuffering from the disease may occur by way of serum analysis forceruloplasmin and iron levels or through MRI analysis of the brain tolook for iron accumulation.

In one embodiment, patients with Aceruloplasminemia may be administereda composition comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA of the present invention. Therare liver disease or disorder polynucleotide, primary construct ormmRNA may encode a peptide, protein or fragment thereof such as, but notlimited to, CP.

In one embodiment, Aceruloplasminemia may be treated by administering acomposition of the present invention comprising at least one rare liverdisease or disorder polynucleotide, primary construct or mmRNA encodinga peptide, protein or fragment thereof of CP. In another embodiment,Aceruloplasminemia may be treated by administering a composition of thepresent invention comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA encoding a peptide, proteinor fragment thereof comprising SEQ ID NO: 891.

Alpha-Mannosidosis

In one embodiment, the rare liver disease or disorder polynucleotide,primary construct, or mmRNA of the present invention may be used totreat alpha-mannosidosis. As used herein, the term “alpha-mannosidosis”refers to a disorder caused by defective and/or dysfunctionalalpha-D-mannosidase enzyme activity resulting in lysosomal storagedisorder. Alpha-mannosidosis, arising from genetic defects in the MAN2B1gene (encoding alpha-D-mannosidase), is an autosomal recessive disorder.Characteristics of those suffering from alpha-mannosidosis include, butare not limited to immunodeficiency, abnormalities of the face andskeleton, impaired hearing and intellectual impairment. Diagnosis mayoccur as early as during infancy using methods known in the artincluding, but not limited to, analysis of phenotypic characteristics aswell as liver and spleen dysfunction. Milder forms of the disease mayremain undiagnosed through childhood and young adulthood. Diagnosis ofthe disease is typically confirmed by measurement of alpha-D-mannosidaseenzymatic activity in while blood cells. (Malm, D. et al.,Alpha-mannosidosis. Orphanet J Rare Dis. 2008 Jul. 23; 3:21; hereinincorporated by reference in its entirety).

In one embodiment, patients with alpha-mannosidosis may be administereda composition comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA of the present invention. Therare liver disease or disorder polynucleotide, primary construct ormmRNA may encode a peptide, protein or fragment thereof such as, but notlimited to, MAN2B1.

In one embodiment, alpha-mannosidosis may be treated by administering acomposition of the present invention comprising at least one rare liverdisease or disorder polynucleotide, primary construct or mmRNA encodinga peptide, protein or fragment thereof of MAN2B1. In another embodiment,alpha-mannosidosis may be treated by administering a composition of thepresent invention comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA encoding a peptide, proteinor fragment thereof comprising SEQ ID NO: 1156-1158.

Tyrosinemia

In one embodiment, the rare liver disease or disorder polynucleotide,primary construct, or mmRNA of the present invention may be used totreat tyrosinemia. As used herein, the term “tyrosinemia” refers to adisorder and/or condition characterized by elevated levels of tyrosineand/or tyrosine byproducts in the blood. In some embodiments,tyrosinemia is caused by a genetic mutation resulting in a defectiveand/or dysfunctional enzyme necessary for tyrosine degradation. Symptomsinclude, but are not limited to failure to thrive, diarrhea, vomiting,jaundice, an increased tendency for nosebleeds, microcephaly, tremor,ataxia, self-mutilating behavior, fine motor coordination disturbances,language deficits, convulsions and/or a cabbage-like odor. (Nakamura, K.et al., Animal models of tyrosinemia. J Nutr. 2007 June; 137(6 Suppl1):15565-15605; discussion 1573S-1575S; Bergeron, A. et al., Hereditarytyrosinemia: an endoplasmic reticulum stress disorder? Med Sci (Paris).2003 October; 19(10):976-80; Mehere, P. et al., Tyrosineaminotransferase: biochemical and structural properties and moleculardynamics simulations. Protein Cell. 2010 November; 1(11):1023-32; eachof which is herein incorporated by reference in its entirety).

Type-1 tyrosinemia refers to a form of the disorder caused by a lack offumarylacetoacetate hydrolase (FAH) activity. The lack of FAH activityin tyrosinemia type-1 leads to an accumulation of the metabolitefumarylacetoacetate, triggering cellular activities such as apoptosis,mutagenesis, aneugenesis and mitogenesis.

Tyrosinemia type-2 refers to a form of the disorder caused by a lack oftyrosine aminotransferase (TAT) activity. TAT is responsible for thereversible transamination of tyrosine as well as other aromatic aminoacids including, but not limited to p-hydroxyphenylpyruvate (pHPP).

In one embodiment, patients with tyrosinemia may be administered acomposition comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA of the present invention. Therare liver disease or disorder polynucleotide, primary construct ormmRNA may encode a peptide, protein or fragment thereof such as, but notlimited to, FAH or TAT.

In one embodiment, tyrosinemia type-1 may be treated by administering acomposition of the present invention comprising at least one rare liverdisease or disorder polynucleotide, primary construct or mmRNA encodinga peptide, protein or fragment thereof of FAH. In another embodiment,tyrosinemia type-1 may be treated by administering a composition of thepresent invention comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA encoding a peptide, proteinor fragment thereof comprising SEQ ID NO: 985-987.

In one embodiment, tyrosinemia type-2 may be treated by administering acomposition of the present invention comprising at least one rare liverdisease or disorder polynucleotide, primary construct or mmRNA encodinga peptide, protein or fragment thereof of TAT. In another embodiment,tyrosinmia type-2 may be treated by administering a composition of thepresent invention comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA encoding a peptide, proteinor fragment thereof comprising SEQ ID NO: 1356.

Hemochromatosis

In one embodiment, the rare liver disease or disorder polynucleotide,primary construct, or mmRNA of the present invention may be used totreat hemochromatosis. As used herein, the term “hemochromatosis” refersto a disorder or condition characterized by iron overload that mayresult from a genetic defect. Symptoms of hemochromatosis include, butare not limited to hepatic cirrhosis, hyperpigmentation, decreasedpituitary function, diabetes and/or arthritis. The genetic defectassociated with hemochromatosis is used to characterize the form of thedisease. Hemochromatosis type 1 is caused by a genetic mutation in theHFE gene. Types 2A and 2B are the result of mutations in the HFE2 andHAMP genes, respectively. While symptoms for hemochromatosis type 1 donot typically present until adulthood, symptoms for hemochromatosistypes 2A and 2B typically present during childhood. (Papanikolaou, G. etal., Hepcidin in iron overload disorders. Blood. 2005 May 15; 105(10):4103-5; Nandar, W. et al., HFE gene variants affect iron in the brain. JNutr. 2011 Apr. 1; 141(4):7295-7395; Wallace, D. F. et al., Non-HFEhaemochromatosis. World J Gastroenterol. 2007 Sep. 21; 13(35):4690-8;each of which is herein incorporated by reference in its entirety).

In one embodiment, patients with hemochromatosis may be administered acomposition comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA of the present invention. Therare liver disease or disorder polynucleotide, primary construct ormmRNA may encode a peptide, protein or fragment thereof such as, but notlimited to, HFE2, HAMP, solute carrier family 40, member 1 (SLC40A1) andtransferrin receptor 2 (TFR2).

In one embodiment, hemochromatosis type-1 may be treated byadministering a composition of the present invention comprising at leastone rare liver disease or disorder polynucleotide, primary construct ormmRNA encoding a peptide, protein or fragment thereof of HFE. In anotherembodiment, hemochromatosis type-1 may be treated by administering acomposition of the present invention comprising at least one rare liverdisease or disorder polynucleotide, primary construct or mmRNA encodinga peptide, protein or fragment thereof comprising SEQ ID NO: 1038-1050.

In one embodiment, hemochromatosis type-2 may be treated byadministering a composition of the present invention comprising at leastone rare liver disease or disorder polynucleotide, primary construct ormmRNA encoding a peptide, protein or fragment thereof of HFE2 or HAMP.In another embodiment, hemochromatosis type-2 may be treated byadministering a composition of the present invention comprising at leastone rare liver disease or disorder polynucleotide, primary construct ormmRNA encoding a peptide, protein or fragment thereof comprising SEQ IDNO: 1051-1057, 1067 or 1068.

In one embodiment, hemochromatosis may be treated by administering acomposition of the present invention comprising at least one rare liverdisease or disorder polynucleotide, primary construct or mmRNA encodinga peptide, protein or fragment thereof of TFR2 or SLC40A1. In anotherembodiment, hemochromatosis may be treated by administering acomposition of the present invention comprising at least one rare liverdisease or disorder polynucleotide, primary construct or mmRNA encodinga peptide, protein or fragment thereof comprising SEQ ID NO: 1290-1296or 1323-1326.

Glycogen Storage Disease

In one embodiment, the rare liver disease or disorder polynucleotide,primary construct, or mmRNA of the present invention may be used totreat glycogen storage disease IV. As used herein, the terms “glycogenstorage disease” or “GSD” refer to a disorder in an organismcharacterized by defective synthesis and/or breakdown of glycogen. Insome embodiments, GSD includes, but is not limited to GSD type 4 alsoknown as Andersen disease, Brancher deficiency, amylopectinosis andglycogen branching enzyme deficiency. GSD type IV results from adeficiency in glycogen branching enzyme (GBE), encoded by the GBE1 gene,leading to the formation of abnormal glycogens that may be cytotoxic.Although variations exist due to the expression of tissue-specificisoforms, patients with classical hepatic GSD type IV appear healthy atbirth; however, cirrhosis of the liver occurs early in life, typicallyleading to liver failure before the 5^(th) year of life. (Ozen, H.,Glycogen storage diseases: new perspectives. World J Gastroenterol. 2007May 14; 13(18):2541-53; herein incorporated by reference in itsentirety).

In one embodiment, patients with GSD may be administered a compositioncomprising at least one rare liver disease or disorder polynucleotide,primary construct or mmRNA of the present invention. The rare liverdisease or disorder polynucleotide, primary construct or mmRNA mayencode a peptide, protein or fragment thereof such as, but not limitedto, glucan (1,4-alpha-), branching enzyme 1 (GBE1).

In one embodiment, GSD type IV may be treated by administering acomposition of the present invention comprising at least one rare liverdisease or disorder polynucleotide, primary construct or mmRNA encodinga peptide, protein or fragment thereof of GBE1. In another embodiment,GSD IV may be treated by administering a composition of the presentinvention comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA encoding a peptide, proteinor fragment thereof comprising SEQ ID NO: 1010-1012.

Fanconi Cystinosis

In one embodiment, the rare liver disease or disorder polynucleotide,primary construct, or mmRNA of the present invention may be used totreat fanconi cystinosis. As used herein, the term “cystinosis” refersto a disease characterized by the abnormal accumulation of cystinewithin lysosomes. It is the most common cause of Fanconi syndrome, akidney disease that prevents reabsorption of metabolites into the bloodstream, causing them to instead be passed in the urine. Cystine isformed through a disulfide bond between two cysteine residues. Lysosomesare sites of hydrolytic protein digestion and cystine accumulatingwithin lysosomes relies on carrier-mediated transport for its release.Cystinosin, encoded by the CTNS gene, is a seven transmembrane proteinthat binds cystine in lysosomes and facilitates its removal fromlysosomes in collaboration with other proteins. Individuals with severedefects in cystinosin function are affected between 6 and 12 months ofage and present with fluid and electrolyte loss as well as other renaland metabolic complications. Renal failure typically occurs by the ageof 10. Current treatment is with the drug cysteamine which is capable ofcleaving cystine molecules, allowing them to be cleared from lysosomes.(Kalatzis, V. et al., Cystinosis: from gene to disease. Nephrol DialTransplant. 2002 November; 17(11):1883-6; herein incorporated byreference in its entirety).

In one embodiment, patients with hemochromatosis may be administered acomposition comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA of the present invention. Therare liver disease or disorder polynucleotide, primary construct ormmRNA may encode a peptide, protein or fragment thereof such as, but notlimited to, cystinosin, lysosomal cysteine transporter (CTNS).

In one embodiment, fanconi cystinosis may be treated by administering acomposition of the present invention comprising at least one rare liverdisease or disorder polynucleotide, primary construct or mmRNA encodinga peptide, protein or fragment thereof of CTNS. In another embodiment,fanconi cystinosis may be treated by administering a composition of thepresent invention comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA encoding a peptide, proteinor fragment thereof comprising SEQ ID NO: 938-941.

Farber Lipogranulomatosis

In one embodiment, the rare liver disease or disorder polynucleotide,primary construct, or mmRNA of the present invention may be used totreat farber lipogranulomatosis. As used herein, the terms “Farberlipogranulomatosis,” “Farber's lipogranulomatosis” or “Farber disease”refers to a lysosomal storage disorder identified by Sidney Farber in1957. The disease is caused by a defect in the enzyme acid ceramindase,encoded by the N-acylsphingosine amidohydrolase (acid ceramidase) 1(ASAH1) gene. Deficiency of this enzyme prevents the normal breakdown ofintracellular ceramide into sphingosine and fatty acid and causes theabnormal accumulation ceramide that leads to disease symptoms. Symptomsare typically present in early infancy, but may also occur in laterlife. In the classic form of the disease, symptoms develop within thefirst few weeks of life and may include deformity of the joints, thepresence of subcutaneous nodules and hoarseness. Patients may alsosuffer neurological impairment, typically leading to death in infancy.Patients with mild neurological impairment may survive into their fourthdecade. Currently, there are no treatments available for Farberlipogranulomatosis. (Ekici, B. et al., Farber disease: A clinicaldiagnosis. J Pediatr Neurosci. 2012 May; 7(2):154-5; Farber, S. et al.,Lipogranulomatosis; a new lipo-glycoprotein storage disease. J Mt SinaiHosp N Y. 1957 November-December; 24(6):816-37; each of which is hereinincorporated by reference in its entirety).

In one embodiment, patients with hemochromatosis may be administered acomposition comprising at least one rare liver disease or disorderpolynucleotide, primary construct or mmRNA of the present invention. Therare liver disease or disorder polynucleotide, primary construct ormmRNA may encode a peptide, protein or fragment thereof such as, but notlimited to, N-acylsphingosine amidohydrolase (acid ceramidase) 1(ASAH1).

In one embodiment, farber lipogranulomatosis may be treated byadministering a composition of the present invention comprising at leastone rare liver disease or disorder polynucleotide, primary construct ormmRNA encoding a peptide, protein or fragment thereof of ASAH1. Inanother embodiment, fanconi cystinosis may be treated by administering acomposition of the present invention comprising at least one rare liverdisease or disorder polynucleotide, primary construct or mmRNA encodinga peptide, protein or fragment thereof comprising SEQ ID NO: 1171-1175.

Wound Management

The polynucleotides, primary constructs or mmRNA of the presentinvention may be used for wound treatment, e.g. of wounds exhibitingdelayed healing. Provided herein are methods comprising theadministration of polynucleotide, primary construct or mmRNA in order tomanage the treatment of wounds. The methods herein may further comprisesteps carried out either prior to, concurrent with or postadministration of the polynucleotide, primary construct or mmRNA. Forexample, the wound bed may need to be cleaned and prepared in order tofacilitate wound healing and hopefully obtain closure of the wound.Several strategies may be used in order to promote wound healing andachieve wound closure including, but not limited to: (i) debridement,optionally repeated, sharp debridement (surgical removal of dead orinfected tissue from a wound), optionally including chemical debridingagents, such as enzymes, to remove necrotic tissue; (ii) wound dressingsto provide the wound with a moist, warm environment and to promotetissue repair and healing.

Examples of materials that are used in formulating wound dressingsinclude, but are not limited to: hydrogels (e.g., AQUASORB®; DUODERM®),hydrocolloids (e.g., AQUACEL®; COMFEEL®), foams (e.g., LYOFOAM®;SPYROSORB®), and alginates (e.g., ALGISITE®; CURASORB®); (iii)additional growth factors to stimulate cell division and proliferationand to promote wound healing e.g. becaplermin (REGRANEX GEL®), a humanrecombinant platelet-derived growth factor that is approved by the FDAfor the treatment of neuropathic foot ulcers; (iv) soft-tissue woundcoverage, a skin graft may be necessary to obtain coverage of clean,non-healing wounds. Examples of skin grafts that may be used forsoft-tissue coverage include, but are not limited to: autologous skingrafts, cadaveric skin graft, bioengineered skin substitutes (e.g.,APLIGRAF®; DERMAGRAFT®).

In certain embodiments, the polynucleotide, primary construct or mmRNAof the present invention may further include hydrogels (e.g., AQUASORB®;DUODERM®), hydrocolloids (e.g., AQUACEL®; COMFEEL®), foams (e.g.,LYOFOAM®; SPYROSORB®), and/or alginates (e.g., ALGISITE®; CURASORB®). Incertain embodiments, the polynucleotide, primary construct or mmRNA ofthe present invention may be used with skin grafts including, but notlimited to, autologous skin grafts, cadaveric skin graft, orbioengineered skin substitutes (e.g., APLIGRAF®; DERMAGRAFT®). In someembodiments, the polynucleotide, primary construct or mmRNA may beapplied with would dressing formulations and/or skin grafts or they maybe applied separately but methods such as, but not limited to, soakingor spraying.

In some embodiments, compositions for wound management may comprise apolynucleotide, primary construct or mmRNA encoding for ananti-microbial polypeptide (e.g., an anti-bacterial polypeptide) and/oran anti-viral polypeptide. A precursor or a partially or fully processedform of the anti-microbial polypeptide may be encoded. The compositionmay be formulated for administration using a bandage (e.g., an adhesivebandage). The anti-microbial polypeptide and/or the anti-viralpolypeptide may be intermixed with the dressing compositions or may beapplied separately, e.g., by soaking or spraying.

Production of Antibodies

In one embodiment of the invention, the polynucleotides, primaryconstructs or mmRNA may encode antibodies and fragments of suchantibodies. These may be produced by any one of the methods describedherein. The antibodies may be of any of the different subclasses orisotypes of immunoglobulin such as, but not limited to, IgA, IgG, orIgM, or any of the other subclasses. Exemplary antibody molecules andfragments that may be prepared according to the invention include, butare not limited to, immunoglobulin molecules, substantially intactimmunoglobulin molecules and those portions of an immunoglobulinmolecule that may contain the paratope. Such portion of antibodies thatcontain the paratope include, but are not limited to Fab, Fab′, F(ab′)₂,F(v) and those portions known in the art.

The polynucleotides of the invention may encode variant antibodypolypeptides which may have a certain identity with a referencepolypeptide sequence, or have a similar or dissimilar bindingcharacteristic with the reference polypeptide sequence.

Antibodies obtained by the methods of the present invention may bechimeric antibodies comprising non-human antibody-derived variableregion(s) sequences, derived from the immunized animals, and humanantibody-derived constant region(s) sequences. In addition, they canalso be humanized antibodies comprising complementary determiningregions (CDRs) of non-human antibodies derived from the immunizedanimals and the framework regions (FRs) and constant regions derivedfrom human antibodies. In another embodiment, the methods providedherein may be useful for enhancing antibody protein product yield in acell culture process.

Managing Infection

In one embodiment, provided are methods for treating or preventing amicrobial infection (e.g., a bacterial infection) and/or a disease,disorder, or condition associated with a microbial or viral infection,or a symptom thereof, in a subject, by administering a polynucleotide,primary construct or mmRNA encoding an anti-microbial polypeptide. Saidadministration may be in combination with an anti-microbial agent (e.g.,an anti-bacterial agent), e.g., an anti-microbial polypeptide or a smallmolecule anti-microbial compound described herein. The anti-microbialagents include, but are not limited to, anti-bacterial agents,anti-viral agents, anti-fungal agents, anti-protozoal agents,anti-parasitic agents, and anti-prion agents.

The agents can be administered simultaneously, for example in a combinedunit dose (e.g., providing simultaneous delivery of both agents). Theagents can also be administered at a specified time interval, such as,but not limited to, an interval of minutes, hours, days or weeks.Generally, the agents may be concurrently bioavailable, e.g.,detectable, in the subject. In some embodiments, the agents may beadministered essentially simultaneously, for example two unit dosagesadministered at the same time, or a combined unit dosage of the twoagents. In other embodiments, the agents may be delivered in separateunit dosages. The agents may be administered in any order, or as one ormore preparations that includes two or more agents. In a preferredembodiment, at least one administration of one of the agents, e.g., thefirst agent, may be made within minutes, one, two, three, or four hours,or even within one or two days of the other agent, e.g., the secondagent. In some embodiments, combinations can achieve synergisticresults, e.g., greater than additive results, e.g., at least 25, 50, 75,100, 200, 300, 400, or 500% greater than additive results.

Conditions Associated with Bacterial Infection

Diseases, disorders, or conditions which may be associated withbacterial infections include, but are not limited to one or more of thefollowing: abscesses, actinomycosis, acute prostatitis, aeromonashydrophila, annual ryegrass toxicity, anthrax, bacillary peliosis,bacteremia, bacterial gastroenteritis, bacterial meningitis, bacterialpneumonia, bacterial vaginosis, bacterium-related cutaneous conditions,bartonellosis, BCG-oma, botryomycosis, botulism, Brazilian purpuricfever, Brodie abscess, brucellosis, Buruli ulcer, campylobacteriosis,caries, Carrion's disease, cat scratch disease, cellulitis, chlamydiainfection, cholera, chronic bacterial prostatitis, chronic recurrentmultifocal osteomyelitis, clostridial necrotizing enteritis, combinedperiodontic-endodontic lesions, contagious bovine pleuropneumonia,diphtheria, diphtheritic stomatitis, ehrlichiosis, erysipelas,piglottitis, erysipelas, Fitz-Hugh-Curtis syndrome, flea-borne spottedfever, foot rot (infectious pododermatitis), Garre's sclerosingosteomyelitis, Gonorrhea, Granuloma inguinale, human granulocyticanaplasmosis, human monocytotropic ehrlichiosis, hundred days' cough,impetigo, late congenital syphilitic oculopathy, legionellosis,Lemierre's syndrome, leprosy (Hansen's Disease), leptospirosis,listeriosis, Lyme disease, lymphadenitis, melioidosis, meningococcaldisease, meningococcal septicaemia, methicillin-resistant Staphylococcusaureus (MRSA) infection, mycobacterium avium-intracellulare (MAI),mycoplasma pneumonia, necrotizing fasciitis, nocardiosis, noma (cancrumoris or gangrenous stomatitis), omphalitis, orbital cellulitis,osteomyelitis, overwhelming post-splenectomy infection (OPSI), ovinebrucellosis, pasteurellosis, periorbital cellulitis, pertussis (whoopingcough), plague, pneumococcal pneumonia, Pott disease, proctitis,pseudomonas infection, psittacosis, pyaemia, pyomyositis, Q fever,relapsing fever (typhinia), rheumatic fever, Rocky Mountain spottedfever (RMSF), rickettsiosis, salmonellosis, scarlet fever, sepsis,serratia infection, shigellosis, southern tick-associated rash illness,staphylococcal scalded skin syndrome, streptococcal pharyngitis,swimming pool granuloma, swine brucellosis, syphilis, syphiliticaortitis, tetanus, toxic shock syndrome (TSS), trachoma, trench fever,tropical ulcer, tuberculosis, tularemia, typhoid fever, typhus,urogenital tuberculosis, urinary tract infections, vancomycin-resistantStaphylococcus aureus infection, Waterhouse-Friderichsen syndrome,pseudotuberculosis (Yersinia) disease, and yersiniosis. Other diseases,disorders, and/or conditions associated with bacterial infections caninclude, for example, Alzheimer's disease, anorexia nervosa, asthma,atherosclerosis, attention deficit hyperactivity disorder, autism,autoimmune diseases, bipolar disorder, cancer (e.g., colorectal cancer,gallbladder cancer, lung cancer, pancreatic cancer, and stomach cancer),chronic fatigue syndrome, chronic obstructive pulmonary disease, Crohn'sdisease, coronary heart disease, dementia, depression, Guillain-Barrésyndrome, metabolic syndrome, multiple sclerosis, myocardial infarction,obesity, obsessive-compulsive disorder, panic disorder, psoriasis,rheumatoid arthritis, sarcoidosis, schizophrenia, stroke,thromboangiitis obliterans (Buerger's disease), and Tourette syndrome.

Bacterial Pathogens

The bacterium described herein can be a Gram-positive bacterium or aGram-negative bacterium. Bacterial pathogens include, but are notlimited to, Acinetobacter baumannii, Bacillus anthracis, Bacillussubtilis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus,Brucella canis, Brucella melitensis, Brucella suis, Campylobacterjejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophilapsittaci, Clostridium botulinum, Clostridium difficile, Clostridiumperfringens, Clostridium tetani, coagulase Negative Staphylococcus,Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium,Escherichia coli, enterotoxigenic Escherichia coli (ETEC),enteropathogenic E. coli, E. coli O157:H7, Enterobacter sp., Francisellatularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiellapneumoniae, Legionella pneumophila, Leptospira interrogans, Listeriamonocytogenes, Moraxella catarralis, Mycobacterium leprae, Mycobacteriumtuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseriameningitides, Preteus mirabilis, Proteus sps., Pseudomonas aeruginosa,Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium,Serratia marcesens, Shigella flexneri, Shigella sonnei, Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus saprophyticus,Streptococcus agalactiae, Streptococcus mutans, Streptococcuspneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae,and Yersinia pestis. Bacterial pathogens may also include bacteria thatcause resistant bacterial infections, for example, clindamycin-resistantClostridium difficile, fluoroquinolon-resistant Clostridium difficile,methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistantEnterococcus faecalis, multidrug-resistant Enterococcus faecium,multidrug-resistance Pseudomonas aeruginosa, multidrug-resistantAcinetobacter baumannii, and vancomycin-resistant Staphylococcus aureus(VRSA).

Antibiotic Combinations

In one embodiment, the modified mRNA of the present invention may beadministered in conjunction with one or more antibiotics. These include,but are not limited to Aknilox, Ambisome, Amoxycillin, Ampicillin,Augmentin, Avelox, Azithromycin, Bactroban, Betadine, Betnovate,Blephamide, Cefaclor, Cefadroxil, Cefdinir, Cefepime, Cefix, Cefixime,Cefoxitin, Cefpodoxime, Cefprozil, Cefuroxime, Cefzil, Cephalexin,Cephazolin, Ceptaz, Chloramphenicol, Chlorhexidine, Chloromycetin,Chlorsig, Ciprofloxacin, Clarithromycin, Clindagel, Clindamycin,Clindatech, Cloxacillin, Colistin, Co-trimoxazole, Demeclocycline,Diclocil, Dicloxacillin, Doxycycline, Duricef, Erythromycin, Flamazine,Floxin, Framycetin, Fucidin, Furadantin, Fusidic, Gatifloxacin,Gemifloxacin, Gemifloxacin, Ilosone, Iodine, Levaquin, Levofloxacin,Lomefloxacin, Maxaquin, Mefoxin, Meronem, Minocycline, Moxifloxacin,Myambutol, Mycostatin, Neosporin, Netromycin, Nitrofurantoin,Norfloxacin, Norilet, Ofloxacin, Omnicef, Ospamox, Oxytetracycline,Paraxin, Penicillin, Pneumovax, Polyfax, Povidone, Rifadin, Rifampin,Rifaximin, Rifinah, Rimactane, Rocephin, Roxithromycin, Seromycin,Soframycin, Sparfloxacin, Staphlex, Targocid, Tetracycline, Tetradox,Tetralysal, tobramycin, Tobramycin, Trecator, Tygacil, Vancocin,Velosef, Vibramycin, Xifaxan, Zagam, Zitrotek, Zoderm, Zymar, and Zyvox.

Antibacterial Agents

Exemplary anti-bacterial agents include, but are not limited to,aminoglycosides (e.g., amikacin (AMIKIN®), gentamicin (GARAMYCIN®),kanamycin (KANTREX®), neomycin (MYCIFRADIN®), netilmicin (NETROMYCIN®),tobramycin (NEBCIN®), Paromomycin (HUMATIN®), ansamycins (e.g.,geldanamycin, herbimycin), carbacephem (e.g., loracarbef (LORABID®),Carbapenems (e.g., ertapenem (INVANZ®), doripenem (DORIBAX®),imipenem/cilastatin (PRIMAXIN®), meropenem (MERREM®), cephalosporins(first generation) (e.g., cefadroxil (DURICEF®), cefazolin (ANCEF®),cefalotin or cefalothin (KEFLIN®), cefalexin (KEFLEX®), cephalosporins(second generation) (e.g., cefaclor (CECLOR®), cefamandole (MANDOL®),cefoxitin (MEFOXIN®), cefprozil (CEFZIL®), cefuroxime (CEFTIN®,ZINNAT®)), cephalosporins (third generation) (e.g., cefixime (SUPRAX®),cefdinir (OMNICEF®, CEFDIEL®), cefditoren (SPECTRACEF®), cefoperazone(CEFOBID®), cefotaxime (CLAFORAN®), cefpodoxime (VANTIN®), ceftazidime(FORTAZ®), ceftibuten (CEDAX®), ceftizoxime (CEFIZOX®), ceftriaxone(ROCEPHIN®)), cephalosporins (fourth generation) (e.g., cefepime(MAXIPIME®)), cephalosporins (fifth generation) (e.g., ceftobiprole(ZEFTERA®)), glycopeptides (e.g., teicoplanin (TARGOCID®), vancomycin(VANCOClN®), telavancin (VIBATIV®)), lincosamides (e.g., clindamycin(CLEOCIN®), lincomycin (LINCOClN®)), lipopeptide (e.g., daptomycin(CUBICIN®)), macrolides (e.g., azithromycin (ZITHROMAX®, SUMAMED®,ZITROCIN®), clarithromycin (BIAXIN®), dirithromycin (DYNABAC®),erythromycin (ERYTHOCIN®, ERYTHROPED®), roxithromycin, troleandomycin(TAO®), telithromycin (KETEK®), spectinomycin (TROBICIN®)), monobactams(e.g., aztreonam (AZACTAM®)), nitrofurans (e.g., furazolidone(FUROXONE®), nitrofurantoin (MACRODANTIN®, MACROBID®)), penicillins(e.g., amoxicillin (NOVAMOX®, AMOXIL®), ampicillin (PRINCIPEN®),azlocillin, carbenicillin (GEOCILLIN®), cloxacillin (TEGOPEN®),dicloxacillin (DYNAPEN®), flucloxacillin (FLOXAPEN®), mezlocillin(MEZLIN®), methicillin (STAPHCILLIN®), nafcillin (UNIPEN®), oxacillin(PROSTAPHLIN®), penicillin G (PENTIDS®), penicillin V (PEN-VEE-K®),piperacillin (PIPRACIL®), temocillin (NEGABAN®), ticarcillin (TICAR®)),penicillin combinations (e.g., amoxicillin/clavulanate (AUGMENTIN®),ampicillin/sulbactam (UNASYN®), piperacillin/tazobactam (ZOSYN®),ticarcillin/clavulanate (TIMENTIN®)), polypeptides (e.g., bacitracin,colistin (COLY-MYCIN-S®), polymyxin B, quinolones (e.g., ciprofloxacin(CIPRO®, CIPROXIN®, CIPROBAY®), enoxacin (PENETREX®), gatifloxacin(TEQUIN®), levofloxacin (LEVAQUIN®), lomefloxacin (MAXAQUIN®),moxifloxacin (AVELOX®), nalidixic acid (NEGGRAM®), norfloxacin(NOROXIN®), ofloxacin (FLOXIN®, OCUFLOX®), trovafloxacin (TROVAN®),grepafloxacin (RAXAR®), sparfloxacin (ZAGAM®), temafloxacin(OMNIFLOX®)), sulfonamides (e.g., mafenide (SULFAMYLON®),sulfonamidochrysoidine (PRONTOSIL®), sulfacetamide (SULAMYD®,BLEPH-100), sulfadiazine (MICRO-SULFON®), silver sulfadiazine(SILVADENE®), sulfamethizole (THIOSULFIL FORTE®), sulfamethoxazole(GANTANOL®), sulfanilimide, sulfasalazine (AZULFIDINE®), sulfisoxazole(GANTRISIN®), trimethoprim (PROLOPRIM®), TRIMPEX®),trimethoprim-sulfamethoxazole (co-trimoxazole) (TMP-SMX) (BACTRIM®,SEPTRA®)), tetracyclines (e.g., demeclocycline (DECLOMYCIN®),doxycycline (VIBRAMYCIN®), minocycline (MINOCIN®), oxytetracycline(TERRAMYCIN®), tetracycline (SUMYCIN®, ACHROMYCIN® V, STECLIN®)), drugsagainst mycobacteria (e.g., clofazimine (LAMPRENE®), dapsone(AVLOSULFON®), capreomycin (CAPASTAT®), cycloserine (SEROMYCIN®),ethambutol (MYAMBUTOL®), ethionamide (TRECATOR®), isoniazid (I.N.H.®),pyrazinamide (ALDINAMIDE®), rifampin (RIFADIN®, RIMACTANE®), rifabutin(MYCOBUTIN®), rifapentine (PRIFTIN®), streptomycin), and others (e.g.,arsphenamine (SALVARSAN®), chloramphenicol (CHLOROMYCETIN®), fosfomycin(MONUROL®), fusidic acid (FUCIDIN®), linezolid (ZYVOX®), metronidazole(FLAGYL®), mupirocin (BACTROBAN®), platensimycin,quinupristin/dalfopristin (SYNERCID®), rifaximin (XIFAXAN®),thiamphenicol, tigecycline (TIGACYL®), timidazole (TINDAMAX®,FASIGYN®)).

Conditions Associated with Viral Infection

In another embodiment, provided are methods for treating or preventing aviral infection and/or a disease, disorder, or condition associated witha viral infection, or a symptom thereof, in a subject, by administeringa polynucleotide, primary construct or mmRNA encoding an anti-viralpolypeptide, e.g., an anti-viral polypeptide described herein incombination with an anti-viral agent, e.g., an anti-viral polypeptide ora small molecule anti-viral agent described herein.

Diseases, disorders, or conditions associated with viral infectionsinclude, but are not limited to, acute febrile pharyngitis,pharyngoconjunctival fever, epidemic keratoconjunctivitis, infantilegastroenteritis, Coxsackie infections, infectious mononucleosis, Burkittlymphoma, acute hepatitis, chronic hepatitis, hepatic cirrhosis,hepatocellular carcinoma, primary HSV-1 infection (e.g.,gingivostomatitis in children, tonsillitis and pharyngitis in adults,keratoconjunctivitis), latent HSV-1 infection (e.g., herpes labialis andcold sores), primary HSV-2 infection, latent HSV-2 infection, asepticmeningitis, infectious mononucleosis, Cytomegalic inclusion disease,Kaposi sarcoma, multicentric Castleman disease, primary effusionlymphoma, AIDS, influenza, Reye syndrome, measles, postinfectiousencephalomyelitis, Mumps, hyperplastic epithelial lesions (e.g., common,flat, plantar and anogenital warts, laryngeal papillomas,epidermodysplasia verruciformis), cervical carcinoma, squamous cellcarcinomas, croup, pneumonia, bronchiolitis, common cold, Poliomyelitis,Rabies, bronchiolitis, pneumonia, influenza-like syndrome, severebronchiolitis with pneumonia, German measles, congenital rubella,Varicella, and herpes zoster.

Viral Pathogens

Viral pathogens include, but are not limited to, adenovirus,coxsackievirus, dengue virus, encephalitis virus, Epstein-Barr virus,hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplexvirus type 1, herpes simplex virus type 2, cytomegalovirus, humanherpesvirus type 8, human immunodeficiency virus, influenza virus,measles virus, mumps virus, human papillomavirus, parainfluenza virus,poliovirus, rabies virus, respiratory syncytial virus, rubella virus,varicella-zoster virus, West Nile virus, and yellow fever virus. Viralpathogens may also include viruses that cause resistant viralinfections.

Antiviral Agents

Exemplary anti-viral agents include, but are not limited to, abacavir(ZIAGEN®), abacavir/lamivudine/zidovudine (trizivir®), aciclovir oracyclovir (CYCLOVIR®, HERPEX®, ACIVIR®, ACIVIRAX®, ZOVIRAX®, ZOVIR®),adefovir (Preveon®, Hepsera®), amantadine (SYMMETREL®), amprenavir(AGENERASE®), ampligen, arbidol, atazanavir (REYATAZ®), boceprevir,cidofovir, darunavir (PREZISTA®), delavirdine (RESCRIPTOR®), didanosine(VIDEX®), docosanol (ABREVA®), edoxudine, efavirenz (SUSTIVA®,STOCRIN®), emtricitabine (EMTRIVA®), emtricitabine/tenofovir/efavirenz(ATRIPLA®), enfuvirtide (FUZEON®), entecavir (BARACLUDE®, ENTAVIR®),famciclovir (FAMVIR®), fomivirsen (VITRAVENE®), fosamprenavir (LEXIVA®,TELZIR®), foscarnet (FOSCAVIR®), fosfonet, ganciclovir (CYTOVENE®,CYMEVENE®, VITRASERT®), GS 9137 (ELVITEGRAVIR®), imiquimod (ALDARA®,ZYCLARA®, BESELNA®), indinavir (CRIXIVAN®), inosine, inosine pranobex(IMUNOVIR®), interferon type I, interferon type II, interferon type III,kutapressin (NEXAVIR®), lamivudine (ZEFFIX®, HEPTOVIR®, EPIVIR®),lamivudine/zidovudine (COMBIVIR®), lopinavir, loviride, maraviroc(SELZENTRY®, CELSENTRI®), methisazone, MK-2048, moroxydine, nelfinavir(VIRACEPT®), nevirapine (VIRAMUNE®), oseltamivir (TAMIFLU®),peginterferon alfa-2a (PEGASYS®), penciclovir (DENAVIR®), peramivir,pleconaril, podophyllotoxin (CONDYLOX®), raltegravir (ISENTRESS®),ribavirin (COPEGUs®, REBETOL®, RIBASPHERE®, VILONA® AND VIRAZOLE®),rimantadine (FLUMADINE®), ritonavir (NORVIR®), pyramidine, saquinavir(INVIRASE®, FORTOVASE®), stavudine, tea tree oil (melaleuca oil),tenofovir (VIREAD®), tenofovir/emtricitabine (TRUVADA®), tipranavir(APTIVUS®), trifluridine (VIROPTIC®), tromantadine (VIRU-MERZ®),valaciclovir (VALTREX®), valganciclovir (VALCYTE®), vicriviroc,vidarabine, viramidine, zalcitabine, zanamivir (RELENZA®), andzidovudine (azidothymidine (AZT), RETROVIR®, RETROVIS®).

Conditions Associated with Fungal Infections

Diseases, disorders, or conditions associated with fungal infectionsinclude, but are not limited to, aspergilloses, blastomycosis,candidasis, coccidioidomycosis, cryptococcosis, histoplasmosis,mycetomas, paracoccidioidomycosis, and tinea pedis. Furthermore, personswith immuno-deficiencies are particularly susceptible to disease byfungal genera such as Aspergillus, Candida, Cryptoccocus, Histoplasma,and Pneumocystis. Other fungi can attack eyes, nails, hair, andespecially skin, the so-called dermatophytic fungi and keratinophilicfungi, and cause a variety of conditions, of which ringworms such asathlete's foot are common. Fungal spores are also a major cause ofallergies, and a wide range of fungi from different taxonomic groups canevoke allergic reactions in some people.

Fungal Pathogens

Fungal pathogens include, but are not limited to, Ascomycota (e.g.,Fusarium oxysporum, Pneumocystis jirovecii, Aspergillus spp.,Coccidioides immitis/posadasii, Candida albicans), Basidiomycota (e.g.,Filobasidiella neoformans, Trichosporon), Microsporidia (e.g.,Encephalitozoon cuniculi, Enterocytozoon bieneusi), and Mucoromycotina(e.g., Mucor circinelloides, Rhizopus oryzae, Lichtheimia corymbifera).

Anti-Fungal Agents

Exemplary anti-fungal agents include, but are not limited to, polyeneantifungals (e.g., natamycin, rimocidin, filipin, nystatin, amphotericinB, candicin, hamycin), imidazole antifungals (e.g., miconazole(MICATIN®, DAKTARIN®), ketoconazole (NIZORAL®, FUNGORAL®, SEBIZOLE®),clotrimazole (LOTRIMIN®, LOTRIMIN® AF, CANESTEN®), econazole,omoconazole, bifonazole, butoconazole, fenticonazole, isoconazole,oxiconazole, sertaconazole (ERTACZO®), sulconazole, tioconazole),triazole antifungals (e.g., albaconazole fluconazole, itraconazole,isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole),thiazole antifungals (e.g., abafungin), allylamines (e.g., terbinafine(LAMISIL®), naftifine (NAFTIN®), butenafine (LOTRIMIN® Ultra)),echinocandins (e.g., anidulafungin, caspofungin, micafungin), and others(e.g., polygodial, benzoic acid, ciclopirox, tolnaftate (TINACTIN®,DESENEX®, AFTATE®), undecylenic acid, flucytosine or 5-fluorocytosine,griseofulvin, haloprogin, sodium bicarbonate, allicin).

Conditions Associated with Protozoal Infection

Diseases, disorders, or conditions associated with protozoal infectionsinclude, but are not limited to, amoebiasis, giardiasis, trichomoniasis,African Sleeping Sickness, American Sleeping Sickness, leishmaniasis(Kala-Azar), balantidiasis, toxoplasmosis, malaria, acanthamoebakeratitis, and babesiosis.

Protozoan Pathogens

Protozoal pathogens include, but are not limited to, Entamoebahistolytica, Giardia lambila, Trichomonas vaginalis, Trypanosoma brucei,T. cruzi, Leishmania donovani, Balantidium coli, Toxoplasma gondii,Plasmodium spp., and Babesia microti.

Anti-Protozoan Agents

Exemplary anti-protozoal agents include, but are not limited to,eflornithine, furazolidone (FUROXONE®, DEPENDAL-M®), melarsoprol,metronidazole (FLAGYL®), ornidazole, paromomycin sulfate (HUMATIN®),pentamidine, pyrimethamine (DARAPRIM®), and tinidazole (TINDAMAX®,FASIGYN®).

Conditions Associated With Parasitic Infection

Diseases, disorders, or conditions associated with parasitic infectionsinclude, but are not limited to, acanthamoeba keratitis, amoebiasis,ascariasis, babesiosis, balantidiasis, baylisascariasis, chagas disease,clonorchiasis, cochliomyia, cryptosporidiosis, diphyllobothriasis,dracunculiasis, echinococcosis, elephantiasis, enterobiasis,fascioliasis, fasciolopsiasis, filariasis, giardiasis, gnathostomiasis,hymenolepiasis, isosporiasis, katayama fever, leishmaniasis, lymedisease, malaria, metagonimiasis, myiasis, onchocerciasis, pediculosis,scabies, schistosomiasis, sleeping sickness, strongyloidiasis,taeniasis, toxocariasis, toxoplasmosis, trichinosis, and trichuriasis.

Parasitic Pathogens

Parasitic pathogens include, but are not limited to, Acanthamoeba,Anisakis, Ascaris lumbricoides, botfly, Balantidium coli, bedbug,Cestoda, chiggers, Cochliomyia hominivorax, Entamoeba histolytica,Fasciola hepatica, Giardia lamblia, hookworm, Leishmania, Linguatulaserrata, liver fluke, Loa boa, Paragonimus, pinworm, Plasmodiumfalciparum, Schistosoma, Strongyloides stercoralis, mite, tapeworm,Toxoplasma gondii, Trypanosoma, whipworm, Wuchereria bancrofti.

Anti-Parasitic Agents

Exemplary anti-parasitic agents include, but are not limited to,antinematodes (e.g., mebendazole, pyrantel pamoate, thiabendazole,diethylcarbamazine, ivermectin), anticestodes (e.g., niclosamide,praziquantel, albendazole), antitrematodes (e.g., praziquantel),antiamoebics (e.g., rifampin, amphotericin B), and antiprotozoals (e.g.,melarsoprol, eflornithine, metronidazole, timidazole).

Conditions Associated with Prion Infection

Diseases, disorders, or conditions associated with prion infectionsinclude, but are not limited to Creutzfeldt-Jakob disease (CJD),iatrogenic Creutzfeldt-Jakob disease (iCJD), variant Creutzfeldt-Jakobdisease (vCJD), familial Creutzfeldt-Jakob disease (fCJD), sporadicCreutzfeldt-Jakob disease (sCJD), Gerstmann-Strä ussler-Scheinkersyndrome (GSS), fatal familial insomnia (FFI), Kuru, Scrapie, bovinespongiform encephalopathy (BSE), mad cow disease, transmissible minkencephalopathy (TME), chronic wasting disease (CWD), feline spongiformencephalopathy (FSE), exotic ungulate encephalopathy (EUE), andspongiform encephalopathy.

Anti-Prion Agents

Exemplary anti-prion agents include, but are not limited to, flupirtine,pentosan polysuphate, quinacrine, and tetracyclic compounds.

Modulation of the Immune Response Avoidance of the Immune Response

As described herein, a useful feature of the polynucleotides, primaryconstructs or mmRNA of the invention is the capacity to reduce, evade oravoid the innate immune response of a cell. In one aspect, providedherein are polynucleotides, primary constructs or mmRNA encoding apolypeptide of interest which when delivered to cells, results in areduced immune response from the host as compared to the responsetriggered by a reference compound, e.g. an unmodified polynucleotidecorresponding to a polynucleotide, primary construct or mmRNA of theinvention, or a different polynucleotide, primary construct or mmRNA ofthe invention. As used herein, a “reference compound” is any molecule orsubstance which when administered to a mammal, results in an innateimmune response having a known degree, level or amount of immunestimmulation. A reference compound need not be a nucleic acid moleculeand it need not be any of the polynucleotides, primary constructs ormmRNA of the invention. Hence, the measure of a polynucleotides, primaryconstructs or mmRNA avoidance, evasion or failure to trigger an immuneresponse can be expressed in terms relative to any compound or substancewhich is known to trigger such a response.

The term “innate immune response” includes a cellular response toexogenous single stranded nucleic acids, generally of viral or bacterialorigin, which involves the induction of cytokine expression and release,particularly the interferons, and cell death. As used herein, the innateimmune response or interferon response operates at the single cell levelcausing cytokine expression, cytokine release, global inhibition ofprotein synthesis, global destruction of cellular RNA, upregulation ofmajor histocompatibility molecules, and/or induction of apoptotic death,induction of gene transcription of genes involved in apoptosis,anti-growth, and innate and adaptive immune cell activation. Some of thegenes induced by type I IFNs include PKR, ADAR (adenosine deaminaseacting on RNA), OAS (2′,5′-oligoadenylate synthetase), RNase L, and Mxproteins. PKR and ADAR lead to inhibition of translation initiation andRNA editing, respectively. OAS is a dsRNA-dependent synthetase thatactivates the endoribonuclease RNase L to degrade ssRNA.

In some embodiments, the innate immune response comprises expression ofa Type I or Type II interferon, and the expression of the Type I or TypeII interferon is not increased more than two-fold compared to areference from a cell which has not been contacted with apolynucleotide, primary construct or mmRNA of the invention.

In some embodiments, the innate immune response comprises expression ofone or more IFN signature genes and where the expression of the one ofmore IFN signature genes is not increased more than three-fold comparedto a reference from a cell which has not been contacted with thepolynucleotide, primary construct or mmRNA of the invention.

While in some circumstances, it might be advantageous to eliminate theinnate immune response in a cell, the invention providespolynucleotides, primary constructs and mmRNA that upon administrationresult in a substantially reduced (significantly less) the immuneresponse, including interferon signaling, without entirely eliminatingsuch a response.

In some embodiments, the immune response is lower by 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% ascompared to the immune response induced by a reference compound. Theimmune response itself may be measured by determining the expression oractivity level of Type 1 interferons or the expression ofinterferon-regulated genes such as the toll-like receptors (e.g., TLR7and TLR8). Reduction of innate immune response can also be measured bymeasuring the level of decreased cell death following one or moreadministrations to a cell population; e.g., cell death is 10%, 25%, 50%,75%, 85%, 90%, 95%, or over 95% less than the cell death frequencyobserved with a reference compound. Moreover, cell death may affectfewer than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or fewer than0.01% of cells contacted with the polynucleotide, primary construct ormmRNA.

In another embodiment, the polynucleotide, primary construct or mmRNA ofthe present invention is significantly less immunogenic than anunmodified in vitro-synthesized RNA molecule polynucleotide, or primaryconstruct with the same sequence or a reference compound. As usedherein, “significantly less immunogenic” refers to a detectable decreasein immunogenicity. In another embodiment, the term refers to a folddecrease in immunogenicity. In another embodiment, the term refers to adecrease such that an effective amount of the polynucleotide, primaryconstruct or mmRNA can be administered without triggering a detectableimmune response. In another embodiment, the term refers to a decreasesuch that the polynucleotide, primary construct or mmRNA can berepeatedly administered without eliciting an immune response sufficientto detectably reduce expression of the recombinant protein. In anotherembodiment, the decrease is such that the polynucleotide, primaryconstruct or mmRNA can be repeatedly administered without eliciting animmune response sufficient to eliminate detectable expression of therecombinant protein.

In another embodiment, the polynucleotide, primary construct or mmRNA is2-fold less immunogenic than its unmodified counterpart or referencecompound. In another embodiment, immunogenicity is reduced by a 3-foldfactor. In another embodiment, immunogenicity is reduced by a 5-foldfactor. In another embodiment, immunogenicity is reduced by a 7-foldfactor. In another embodiment, immunogenicity is reduced by a 10-foldfactor. In another embodiment, immunogenicity is reduced by a 15-foldfactor. In another embodiment, immunogenicity is reduced by a foldfactor. In another embodiment, immunogenicity is reduced by a 50-foldfactor. In another embodiment, immunogenicity is reduced by a 100-foldfactor. In another embodiment, immunogenicity is reduced by a 200-foldfactor. In another embodiment, immunogenicity is reduced by a 500-foldfactor. In another embodiment, immunogenicity is reduced by a 1000-foldfactor. In another embodiment, immunogenicity is reduced by a 2000-foldfactor. In another embodiment, immunogenicity is reduced by another folddifference.

Methods of determining immunogenicity are well known in the art, andinclude, e.g. measuring secretion of cytokines (e.g. IL-12, IFNalpha,TNF-alpha, RANTES, MIP-1alpha or beta, IL-6, IFN-beta, or IL-8),measuring expression of DC activation markers (e.g. CD83, HLA-DR, CD80and CD86), or measuring ability to act as an adjuvant for an adaptiveimmune response.

The polynucleotide, primary construct or mmRNA of the invention,including the combination of modifications taught herein may havesuperior properties making them more suitable as therapeutic modalities.

It has been determined that the “all or none” model in the art is sorelyinsufficient to describe the biological phenomena associated with thetherapeutic utility of modified mRNA. The present inventors havedetermined that to improve protein production, one may consider thenature of the modification, or combination of modifications, the percentmodification and survey more than one cytokine or metric to determinethe efficacy and risk profile of a particular modified mRNA.

In one aspect of the invention, methods of determining the effectivenessof a modified mRNA as compared to unmodified involves the measure andanalysis of one or more cytokines whose expression is triggered by theadministration of the exogenous nucleic acid of the invention. Thesevalues are compared to administration of an umodified nucleic acid or toa standard metric such as cytokine response, PolylC, R-848 or otherstandard known in the art.

One example of a standard metric developed herein is the measure of theratio of the level or amount of encoded polypeptide (protein) producedin the cell, tissue or organism to the level or amount of one or more(or a panel) of cytokines whose expression is triggered in the cell,tissue or organism as a result of administration or contact with themodified nucleic acid. Such ratios are referred to herein as theProtein:Cytokine Ratio or “PC” Ratio. The higher the PC ratio, the moreefficacioius the modified nucleic acid (polynucleotide encoding theprotein measured). Preferred PC Ratios, by cytokine, of the presentinvention may be greater than 1, greater than 10, greater than 100,greater than 1000, greater than 10,000 or more. Modified nucleic acidshaving higher PC Ratios than a modified nucleic acid of a different orunmodified construct are preferred.

The PC ratio may be further qualified by the percent modificationpresent in the polynucleotide. For example, normalized to a 100%modified nucleic acid, the protein production as a function of cytokine(or risk) or cytokine profile can be determined.

In one embodiment, the present invention provides a method fordetermining, across chemistries, cytokines or percent modification, therelative efficacy of any particular modified the polynucleotide, primaryconstruct or mmRNA by comparing the PC Ratio of the modified nucleicacid (polynucleotide, primary construct or mmRNA).

mmRNA containing varying levels of nucleobase substitutions could beproduced that maintain increased protein production and decreasedimmunostimulatory potential. The relative percentage of any modifiednucleotide to its naturally occurring nucleotide counterpart can bevaried during the IVT reaction (for instance, 100, 50, 25, 10, 5, 2.5,1, 0.1, 0.01% 5 methyl cytidine usage versus cytidine; 100, 50, 25, 10,5, 2.5, 1, 0.1, 0.01% pseudouridine or N1-methyl-pseudouridine usageversus uridine). mmRNA can also be made that utilize different ratiosusing 2 or more different nucleotides to the same base (for instance,different ratios of pseudouridine and N1-methyl-pseudouridine). mmRNAcan also be made with mixed ratios at more than 1 “base” position, suchas ratios of 5 methyl cytidine/cytidine andpseudouridine/N1-methyl-pseudouridine/uridine at the same time. Use ofmodified mRNA with altered ratios of modified nucleotides can bebeneficial in reducing potential exposure to chemically modifiednucleotides. Lastly, positional introduction of modified nucleotidesinto the mmRNA which modulate either protein production orimmunostimulatory potential or both is also possible. The ability ofsuch mmRNA to demonstrate these improved properties can be assessed invitro (using assays such as the PBMC assay described herein), and canalso be assessed in vivo through measurement of both mmRNA-encodedprotein production and mediators of innate immune recognition such ascytokines.

In another embodiment, the relative immunogenicity of thepolynucleotide, primary construct or mmRNA and its unmodifiedcounterpart are determined by determining the quantity of thepolynucleotide, primary construct or mmRNA required to elicit one of theabove responses to the same degree as a given quantity of the unmodifiednucleotide or reference compound. For example, if twice as muchpolynucleotide, primary construct or mmRNA is required to elicit thesame response, than the polynucleotide, primary construct or mmRNA istwo-fold less immunogenic than the unmodified nucleotide or thereference compound.

In another embodiment, the relative immunogenicity of thepolynucleotide, primary construct or mmRNA and its unmodifiedcounterpart are determined by determining the quantity of cytokine (e.g.IL-12, IFNalpha, TNF-alpha, RANTES, MIP-1alpha or beta, IL-6, IFN-beta,or IL-8) secreted in response to administration of the polynucleotide,primary construct or mmRNA, relative to the same quantity of theunmodified nucleotide or reference compound. For example, if one-half asmuch cytokine is secreted, than the polynucleotide, primary construct ormmRNA is two-fold less immunogenic than the unmodified nucleotide. Inanother embodiment, background levels of stimulation are subtractedbefore calculating the immunogenicity in the above methods.

Provided herein are also methods for performing the titration, reductionor elimination of the immune response in a cell or a population ofcells. In some embodiments, the cell is contacted with varied doses ofthe same polynucleotides, primary constructs or mmRNA and dose responseis evaluated. In some embodiments, a cell is contacted with a number ofdifferent polynucleotides, primary constructs or mmRNA at the same ordifferent doses to determine the optimal composition for producing thedesired effect. Regarding the immune response, the desired effect may beto avoid, evade or reduce the immune response of the cell. The desiredeffect may also be to alter the efficiency of protein production.

The polynucleotides, primary constructs and/or mmRNA of the presentinvention may be used to reduce the immune response using the methoddescribed in International Publication No. WO2013003475, hereinincorporated by reference in its entirety.

Activation of the immune response: Vaccines

Additionally, certain modified nucleosides, or combinations thereof,when introduced into the polynucleotides, primary constructs or mmRNA ofthe invention will activate the innate immune response. Such activatingmolecules are useful as adjuvants when combined with polypeptides and/orother vaccines. In certain embodiments, the activating molecules containa translatable region which encodes for a polypeptide sequence useful asa vaccine, thus providing the ability to be a self-adjuvant.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAof the invention may encode an immunogen. The delivery of thepolynucleotides, primary constructs and/or mmRNA encoding an immunogenmay activate the immune response. As a non-limiting example, thepolynucleotides, primary constructs and/or mmRNA encoding an immunogenmay be delivered to cells to trigger multiple innate response pathways(see International Pub. No. WO2012006377; herein incorporated byreference in its entirety). As another non-limiting example, thepolynucleotides, primary constructs and mmRNA of the present inventionencoding an immunogen may be delivered to a vertebrate in a dose amountlarge enough to be immunogenic to the vertebrate (see International Pub.No. WO2012006372 and WO2012006369; each of which is herein incorporatedby reference in their entirety).

The polynucleotides, primary constructs or mmRNA of invention may encodea polypeptide sequence for a vaccine and may further comprise aninhibitor. The inhibitor may impair antigen presentation and/or inhibitvarious pathways known in the art. As a non-limiting example, thepolynucleotides, primary constructs or mmRNA of the invention may beused for a vaccine in combination with an inhibitor which can impairantigen presentation (see International Pub. No. WO2012089225 andWO2012089338; each of which is herein incorporated by reference in theirentirety).

In one embodiment, the polynucleotides, primary constructs or mmRNA ofthe invention may be self-replicating RNA. Self-replicating RNAmolecules can enhance efficiency of RNA delivery and expression of theenclosed gene product. In one embodiment, the polynucleotides, primaryconstructs or mmRNA may comprise at least one modification describedherein and/or known in the art. In one embodiment, the self-replicatingRNA can be designed so that the self-replicating RNA does not induceproduction of infectious viral particles. As a non-limiting example theself-replicating RNA may be designed by the methods described in US Pub.No. US20110300205 and International Pub. No. WO2011005799, each of whichis herein incorporated by reference in their entirety.

In one embodiment, the self-replicating polynucleotides, primaryconstructs or mmRNA of the invention may encode a protein which mayraise the immune response. As a non-limiting example, thepolynucleotides, primary constructs or mmRNA may be self-replicatingmRNA may encode at least one antigen (see US Pub. No. US20110300205 andInternational Pub. Nos. WO2011005799, WO2013006838 and WO2013006842;each of which is herein incorporated by reference in their entirety).

In one embodiment, the self-replicating polynucleotides, primaryconstructs or mmRNA of the invention may be formulated using methodsdescribed herein or known in the art. As a non-limiting example, theself-replicating RNA may be formulated for delivery by the methodsdescribed in Geall et al (Nonviral delivery of self-amplifying RNAvaccines, PNAS 2012; PMID: 22908294).

In one embodiment, the polynucleotides, primary constructs or mmRNA ofthe present invention may encode amphipathic and/or immunogenicamphipathic peptides.

In on embodiment, a formulation of the polynucleotides, primaryconstructs or mmRNA of the present invention may further comprise anamphipathic and/or immunogenic amphipathic peptide. As a non-limitingexample, the polynucleotides, primary constructs or mmRNA comprising anamphipathic and/or immunogenic amphipathic peptide may be formulated asdescribed in US. Pub. No. US20110250237 and International Pub. Nos.WO2010009277 and WO2010009065; each of which is herein incorporated byreference in their entirety.

In one embodiment, the polynucleotides, primary constructs or mmRNA ofthe present invention may be immunostimultory. As a non-limitingexample, the polynucleotides, primary constructs or mmRNA may encode allor a part of a positive-sense or a negative-sense stranded RNA virusgenome (see International Pub No. WO2012092569 and US Pub No.US20120177701, each of which is herein incorporated by reference intheir entirety). In another non-limiting example, the immunostimultorypolynucleotides, primary constructs or mmRNA of the present inventionmay be formulated with an excipient for administration as describedherein and/or known in the art (see International Pub No. WO2012068295and US Pub No. US20120213812, each of which is herein incorporated byreference in their entirety).

In one embodiment, the response of the vaccine formulated by the methodsdescribed herein may be enhanced by the addition of various compounds toinduce the therapeutic effect. As a non-limiting example, the vaccineformulation may include a MHC II binding peptide or a peptide having asimilar sequence to a MHC II binding peptide (see International Pub Nos.WO2012027365, WO2011031298 and US Pub No. US20120070493, US20110110965,each of which is herein incorporated by reference in their entirety). Asanother example, the vaccine formulations may comprise modifiednicotinic compounds which may generate an antibody response to nicotineresidue in a subject (see International Pub No. WO2012061717 and US PubNo. US20120114677, each of which is herein incorporated by reference intheir entirety).

Naturally Occurring Mutants

In another embodiment, the polynucleotides, primary construct and/ormmRNA can be utilized to express variants of naturally occurringproteins that have an improved disease modifying activity, includingincreased biological activity, improved patient outcomes, or aprotective function, etc. Many such modifier genes have been describedin mammals (Nadeau, Current Opinion in Genetics & Development 200313:290-295; Hamilton and Yu, PLoS Genet. 2012; 8:e1002644; Corder etal., Nature Genetics 1994 7:180-184; all herein incorporated byreference in their entireties). Examples in humans include Apo E2protein, Apo A-I variant proteins (Apo A-I Milano, Apo A-I Paris),hyperactive Factor IX protein (Factor IX Padua Arg338Lys), transthyretinmutants (TTR Thr119Met). Expression of ApoE2 (cys112, cys158) has beenshown to confer protection relative to other ApoE isoforms (ApoE3 (cyst12, arg158), and ApoE4 (arg112, arg158)) by reducing susceptibility toAlzheimer's disease and possibly other conditions such as cardiovasculardisease (Corder et al., Nature Genetics 1994 7:180-184; Seripa et al.,Rejuvenation Res. 2011 14:491-500; Liu et al. Nat Rev Neurol. 20139:106-118; all herein incorporated by reference in their entireties).Expression of Apo A-I variants has been associated with reducedcholesterol (deGoma and Rader, 2011 Nature Rev Cardiol 8:266-271; Nissenet al., 2003 JAMA 290:2292-2300; all herein incorporated by reference inits entirety). The amino acid sequence of ApoA-I in certain populationshas been changed to cysteine in Apo A-I Milano (Arg 173 changed to Cys)and in Apo A-I Paris (Arg 151 changed to Cys). Factor IX mutation atposition R338L (FIX Padua) results in a Factor IX protein that has˜10-fold increased activity (Simioni et al., N Engl J. Med. 2009361:1671-1675; Finn et al., Blood. 2012 120:4521-4523; Cantore et al.,Blood. 2012 120:4517-20; all herein incorporated by reference in theirentireties). Mutation of transthyretin at positions 104 or 119 (Arg104 His, Thr119Met) has been shown to provide protection to patients alsoharboring the disease causing Val30Met mutations (Saraiva, Hum Mutat.2001 17:493-503; DATA BASE ON TRANSTHYRETIN MUTATIONShttp://www.ibmc.up.pt/mjsaraiva/ttrmut.html; all herein incorporated byreference in its entirety). Differences in clinical presentation andseverity of symptoms among Portuguese and Japanese Met 30 patientscarrying respectively the Met 119 and the H is 104 mutations areobserved with a clear protective effect exerted by the non pathogenicmutant (Coelho et al. 1996 Neuromuscular Disorders (Suppl) 6: S20;Terazaki et al. 1999. Biochem Biophys Res Commun 264: 365-370; allherein incorporated by reference in its entirety), which confer morestability to the molecule. A modified mRNA encoding these protective TTRalleles can be expressed in TTR amyloidosis patients, thereby reducingthe effect of the pathogenic mutant TTR protein.

Major Groove Interacting Partners

As described herein, the phrase “major groove interacting partner”refers to RNA recognition receptors that detect and respond to RNAligands through interactions, e.g. binding, with the major groove faceof a nucleotide or nucleic acid. As such, RNA ligands comprisingmodified nucleotides or nucleic acids such as the polynucleotide,primary construct or mmRNA as described herein decrease interactionswith major groove binding partners, and therefore decrease an innateimmune response.

Example major groove interacting, e.g. binding, partners include, butare not limited to the following nucleases and helicases. Withinmembranes, TLRs (Toll-like Receptors) 3, 7, and 8 can respond to single-and double-stranded RNAs. Within the cytoplasm, members of thesuperfamily 2 class of DEX(D/H) helicases and ATPases can sense RNAs toinitiate antiviral responses. These helicases include the RIG-I(retinoic acid-inducible gene I) and MDA5 (melanomadifferentiation-associated gene 5). Other examples include laboratory ofgenetics and physiology 2 (LGP2), HIN-200 domain containing proteins, orHelicase-domain containing proteins.

Targeting Of Pathogenic Organisms Or Diseased Cells

Provided herein are methods for targeting pathogenic microorganisms,such as bacteria, yeast, protozoa, helminthes and the like, or diseasedcells such as cancer cells using polynucleotides, primary constructs ormmRNA that encode cytostatic or cytotoxic polypeptides. Preferably themRNA introduced contains modified nucleosides or other nucleic acidsequence modifications that are translated exclusively, orpreferentially, in the target pathogenic organism, to reduce possibleoff-target effects of the therapeutic. Such methods are useful forremoving pathogenic organisms or killing diseased cells found in anybiological material, including blood, semen, eggs, and transplantmaterials including embryos, tissues, and organs.

Bioprocessing

The methods provided herein may be useful for enhancing protein productyield in a cell culture process. In a cell culture containing aplurality of host cells, introduction of a polynucleotide, primaryconstruct or mmRNA described herein results in increased proteinproduction efficiency relative to a corresponding unmodified nucleicacid. Such increased protein production efficiency can be demonstrated,e.g., by showing increased cell transfection, increased proteintranslation from the polynucleotide, primary construct or mmRNA,decreased nucleic acid degradation, and/or reduced innate immuneresponse of the host cell. Protein production can be measured byenzyme-linked immunosorbent assay (ELISA), and protein activity can bemeasured by various functional assays known in the art. The proteinproduction may be generated in a continuous or a batch-fed mammalianprocess.

Additionally, it is useful to optimize the expression of a specificpolypeptide in a cell line or collection of cell lines of potentialinterest, particularly a polypeptide of interest such as a proteinvariant of a reference protein having a known activity. In oneembodiment, provided is a method of optimizing expression of apolypeptide of interest in a target cell, by providing a plurality oftarget cell types, and independently contacting with each of theplurality of target cell types a polynucleotide, primary construct ormmRNA encoding a polypeptide of interest. The cells may be transfectedwith two or more polynucleotide, primary construct or mmRNAsimultaneously or sequentially.

In certain embodiments, multiple rounds of the methods described hereinmay be used to obtain cells with increased expression of one or morenucleic acids or proteins of interest. For example, cells may betransfected with one or more polynucleotide, primary construct or mmRNAthat encode a nucleic acid or protein of interest. The cells may beisolated according to methods described herein before being subjected tofurther rounds of transfections with one or more other nucleic acidswhich encode a nucleic acid or protein of interest before being isolatedagain. This method may be useful for generating cells with increasedexpression of a complex of proteins, nucleic acids or proteins in thesame or related biological pathway, nucleic acids or proteins that actupstream or downstream of each other, nucleic acids or proteins thathave a modulating, activating or repressing function to each other,nucleic acids or proteins that are dependent on each other for functionor activity, or nucleic acids or proteins that share homology.

Additionally, culture conditions may be altered to increase proteinproduction efficiency. Subsequently, the presence and/or level of thepolypeptide of interest in the plurality of target cell types isdetected and/or quantitated, allowing for the optimization of apolypeptide's expression by selection of an efficient target cell andcell culture conditions relating thereto. Such methods are particularlyuseful when the polypeptide contains one or more post-translationalmodifications or has substantial tertiary structure, situations whichoften complicate efficient protein production.

In one embodiment, the cells used in the methods of the presentinvention may be cultured. The cells may be cultured in suspension or asadherent cultures. The cells may be cultured in a varied of vesselsincluding, but not limited to, bioreactors, cell bags, wave bags,culture plates, flasks and other vessels well known to those of ordinaryskill in the art. Cells may be cultured in IMDM (Invitrogen, Catalognumber 12440-53) or any other suitable media including, but not limitedto, chemically defined media formulations. The ambient conditions whichmay be suitable for cell culture, such as temperature and atmosphericcomposition, are well known to those skilled in the art. The methods ofthe invention may be used with any cell that is suitable for use inprotein production.

The invention provides for the repeated introduction (e.g.,transfection) of modified nucleic acids into a target cell population,e.g., in vitro, ex vivo, in situ, or in vivo. For example, contactingthe same cell population may be repeated one or more times (such as two,three, four, five or more than five times). In some embodiments, thestep of contacting the cell population with the polynucleotides, primaryconstructs or mmRNA is repeated a number of times sufficient such that apredetermined efficiency of protein translation in the cell populationis achieved. Given the often reduced cytotoxicity of the target cellpopulation provided by the nucleic acid modifications, repeatedtransfections are achievable in a diverse array of cell types and withina variety of tissues, as provided herein.

In one embodiment, the bioprocessing methods of the present inventionmay be used to produce antibodies or functional fragments thereof. Thefunctional fragments may comprise a Fab, Fab′, F(ab′)₂, an Fv domain, anscFv, or a diabody. They may be variable in any region including thecomplement determining region (CDR). In one embodiment, there iscomplete diversity in the CDR3 region. In another embodiment, theantibody is substantially conserved except in the CDR3 region.

Antibodies may be made which bind or associate with any biomolecule,whether human, pathogenic or non-human in origin. The pathogen may bepresent in a non-human mammal, a clinical specimen or from a commercialproduct such as a cosmetic or pharmaceutical material. They may alsobind to any specimen or sample including clinical specimens or tissuesamples from any organism.

In some embodiments, the contacting step is repeated multiple times at afrequency selected from the group consisting of: 6 hour, 12 hour, 24hour, 36 hour, 48 hour, 72 hour, 84 hour, 96 hour, and 108 hour and atconcentrations of less than 20 nM, less than 50 nM, less than 80 nM orless than 100 nM. Compositions may also be administered at less than 1mM, less than 5 mM, less than 10 mM, less than 100 mM or less than 500mM.

In some embodiments, the polynucleotides, primary constructs or mmRNAare added at an amount of 50 molecules per cell, 100 molecules/cell, 200molecules/cell, 300 molecules/cell, 400 molecules/cell, 500molecules/cell, 600 molecules/cell, 700 molecules/cell, 800molecules/cell, 900 molecules/cell, 1000 molecules/cell, 2000molecules/cell, or 5000 molecules/cell.

In other embodiments, the polynucleotides, primary constructs or mmRNAare added at a concentration selected from the group consisting of: 0.01fmol/106 cells, 0.1 fmol/106 cells, 0.5 fmol/106 cells, 0.75 fmol/106cells, 1 fmol/106 cells, 2 fmol/106 cells, 5 fmol/106 cells, 10 fmol/106cells, 20 fmol/106 cells, 30 fmol/106 cells, 40 fmol/106 cells, 50fmol/106 cells, 60 fmol/106 cells, 100 fmol/106 cells, 200 fmol/106cells, 300 fmol/106 cells, 400 fmol/106 cells, 500 fmol/106 cells, 700fmol/106 cells, 800 fmol/106 cells, 900 fmol/106 cells, and 1 pmol/106cells.

In some embodiments, the production of a biological product upon isdetected by monitoring one or more measurable bioprocess parameters,such as a parameter selected from the group consisting of: cell density,pH, oxygen levels, glucose levels, lactic acid levels, temperature, andprotein production. Protein production can be measured as specificproductivity (SP) (the concentration of a product, such as aheterologously expressed polypeptide, in solution) and can be expressedas mg/L or g/L; in the alternative, specific productivity can beexpressed as pg/cell/day. An increase in SP can refer to an absolute orrelative increase in the concentration of a product produced under twodefined set of conditions (e.g., when compared with controls not treatedwith modified mRNA(s)).

Cells

In one embodiment, the cells are selected from the group consisting ofmammalian cells, bacterial cells, plant, microbial, algal and fungalcells. In some embodiments, the cells are mammalian cells, such as, butnot limited to, human, mouse, rat, goat, horse, rabbit, hamster or cowcells. In a further embodiment, the cells may be from an establishedcell line, including, but not limited to, HeLa, NS0, SP2/0, KEK 293T,Vero, Caco, Caco-2, MDCK, COS-1, COS-7, K562, Jurkat, CHO-K1, DG44,CHOK1SV, CHO-S, Huvec, CV-1, Huh-7, NIH3T3, HEK293, 293, A549, HepG2,IMR-90, MCF-7, U-20S, Per.C6, SF9, SF21 or Chinese Hamster Ovary (CHO)cells.

In certain embodiments, the cells are fungal cells, such as, but notlimited to, Chrysosporium cells, Aspergillus cells, Trichoderma cells,Dictyostelium cells, Candida cells, Saccharomyces cells,Schizosaccharomyces cells, and Penicillium cells.

In certain embodiments, the cells are bacterial cells such as, but notlimited to, E. coli, B. subtilis, or BL21 cells. Primary and secondarycells to be transfected by the methods of the invention can be obtainedfrom a variety of tissues and include, but are not limited to, all celltypes which can be maintained in culture. For examples, primary andsecondary cells which can be transfected by the methods of the inventioninclude, but are not limited to, fibroblasts, keratinocytes, epithelialcells (e.g., mammary epithelial cells, intestinal epithelial cells),endothelial cells, glial cells, neural cells, formed elements of theblood (e.g., lymphocytes, bone marrow cells), muscle cells andprecursors of these somatic cell types. Primary cells may also beobtained from a donor of the same species or from another species (e.g.,mouse, rat, rabbit, cat, dog, pig, cow, bird, sheep, goat, horse).

Purification and Isolation

Those of ordinary skill in the art should be able to make adetermination of the methods to use to purify or isolate of a protein ofinterest from cultured cells. Generally, this is done through a capturemethod using affinity binding or non-affinity purification. If theprotein of interest is not secreted by the cultured cells, then a lysisof the cultured cells should be performed prior to purification orisolation. One may use unclarified cell culture fluid containing theprotein of interest along with cell culture media components as well ascell culture additives, such as anti-foam compounds and other nutrientsand supplements, cells, cellular debris, host cell proteins, DNA,viruses and the like in the present invention. The process may beconducted in the bioreactor itself. The fluid may either bepreconditioned to a desired stimulus such as pH, temperature or otherstimulus characteristic or the fluid can be conditioned upon theaddition of polymer(s) or the polymer(s) can be added to a carrierliquid that is properly conditioned to the required parameter for thestimulus condition required for that polymer to be solubilized in thefluid. The polymer may be allowed to circulate thoroughly with the fluidand then the stimulus may be applied (change in pH, temperature, saltconcentration, etc) and the desired protein and polymer(s) precipitatecan out of the solution. The polymer and the desired protein(s) can beseparated from the rest of the fluid and optionally washed one or moretimes to remove any trapped or loosely bound contaminants. The desiredprotein may then be recovered from the polymer(s) by, for example,elution and the like. Preferably, the elution may be done under a set ofconditions such that the polymer remains in its precipitated form andretains any impurities to it during the selected elution of the desiredprotein. The polymer and protein as well as any impurities may besolubilized in a new fluid such as water or a buffered solution and theprotein may be recovered by a means such as affinity, ion exchanged,hydrophobic, or some other type of chromatography that has a preferenceand selectivity for the protein over that of the polymer or impurities.The eluted protein may then be recovered and may be subjected toadditional processing steps, either batch like steps or continuous flowthrough steps if appropriate.

In another embodiment, it may be useful to optimize the expression of aspecific polypeptide in a cell line or collection of cell lines ofpotential interest, particularly a polypeptide of interest such as aprotein variant of a reference protein having a known activity. In oneembodiment, provided is a method of optimizing expression of apolypeptide of interest in a target cell, by providing a plurality oftarget cell types, and independently contacting with each of theplurality of target cell types a modified mRNA encoding a polypeptide.Additionally, culture conditions may be altered to increase proteinproduction efficiency. Subsequently, the presence and/or level of thepolypeptide of interest in the plurality of target cell types isdetected and/or quantitated, allowing for the optimization of apolypeptide of interest's expression by selection of an efficient targetcell and cell culture conditions relating thereto. Such methods may beuseful when the polypeptide of interest contains one or morepost-translational modifications or has substantial tertiary structure,which often complicate efficient protein production.

Protein Recovery

The protein of interest may be preferably recovered from the culturemedium as a secreted polypeptide, or it can be recovered from host celllysates if expressed without a secretory signal. It may be necessary topurify the protein of interest from other recombinant proteins and hostcell proteins in a way that substantially homogenous preparations of theprotein of interest are obtained. The cells and/or particulate celldebris may be removed from the culture medium or lysate. The product ofinterest may then be purified from contaminant soluble proteins,polypeptides and nucleic acids by, for example, fractionation onimmunoaffinity or ion-exchange columns, ethanol precipitation, reversephase HPLC(RP-HPLC), SEPHADEX® chromatography, chromatography on silicaor on a cation exchange resin such as DEAE. Methods of purifying aprotein heterologous expressed by a host cell are well known in the art.

Methods and compositions described herein may be used to produceproteins which are capable of attenuating or blocking the endogenousagonist biological response and/or antagonizing a receptor or signalingmolecule in a mammalian subject. For example, IL-12 and IL-23 receptorsignaling may be enhanced in chronic autoimmune disorders such asmultiple sclerosis and inflammatory diseases such as rheumatoidarthritis, psoriasis, lupus erythematosus, ankylosing spondylitis andChron's disease (Kikly K, Liu L, Na S, Sedgwich JD (2006) Cur. Opin.Immunol. 18(6): 670-5). In another embodiment, a nucleic acid encodes anantagonist for chemokine receptors. Chemokine receptors CXCR-4 and CCR-5are required for HIV enry into host cells (Arenzana-Seisdedos F et al,(1996) Nature. October 3; 383 (6599):400).

Gene Silencing

The polynucleotides, primary constructs and mmRNA described herein areuseful to silence (i.e., prevent or substantially reduce) expression ofone or more target genes in a cell population. A polynucleotide, primaryconstruct or mmRNA encoding a polypeptide of interest capable ofdirecting sequence-specific histone H3 methylation is introduced intothe cells in the population under conditions such that the polypeptideis translated and reduces gene transcription of a target gene viahistone H3 methylation and subsequent heterochromatin formation. In someembodiments, the silencing mechanism is performed on a cell populationpresent in a mammalian subject. By way of non-limiting example, a usefultarget gene is a mutated Janus Kinase-2 family member, wherein themammalian subject expresses the mutant target gene suffers from amyeloproliferative disease resulting from aberrant kinase activity.

Co-administration of polynucleotides, primary constructs and mmRNA andRNAi agents are also provided herein.

Modulation of Biological Pathways

The rapid translation polynucleotides, primary constructs and mmRNAintroduced into cells provides a desirable mechanism of modulatingtarget biological pathways. Such modulation includes antagonism oragonism of a given pathway. In one embodiment, a method is provided forantagonizing a biological pathway in a cell by contacting the cell withan effective amount of a composition comprising a polynucleotide,primary construct or mmRNA encoding a polypeptide of interest, underconditions such that the polynucleotides, primary constructs and mmRNAis localized into the cell and the polypeptide is capable of beingtranslated in the cell from the polynucleotides, primary constructs andmmRNA, wherein the polypeptide inhibits the activity of a polypeptidefunctional in the biological pathway. Exemplary biological pathways arethose defective in an autoimmune or inflammatory disorder such asmultiple sclerosis, rheumatoid arthritis, psoriasis, lupuserythematosus, ankylosing spondylitis colitis, or Crohn's disease; inparticular, antagonism of the IL-12 and IL-23 signaling pathways are ofparticular utility. (See Kikly K, Liu L, Na S, Sedgwick JD (2006) Curr.Opin. Immunol. 18 (6): 670-5).

Further, provided are polynucleotide, primary construct or mmRNAencoding an antagonist for chemokine receptors; chemokine receptorsCXCR-4 and CCR-5 are required for, e.g., HIV entry into host cells(Arenzana-Seisdedos F et al, (1996) Nature. October 3; 383(6599):400).

Alternatively, provided are methods of agonizing a biological pathway ina cell by contacting the cell with an effective amount of apolynucleotide, primary construct or mmRNA encoding a recombinantpolypeptide under conditions such that the nucleic acid is localizedinto the cell and the recombinant polypeptide is capable of beingtranslated in the cell from the nucleic acid, and the recombinantpolypeptide induces the activity of a polypeptide functional in thebiological pathway. Exemplary agonized biological pathways includepathways that modulate cell fate determination. Such agonization isreversible or, alternatively, irreversible.

Expression of Ligand or Receptor on Cell Surface

In some aspects and embodiments of the aspects described herein, thepolynucleotides, primary constructs or mmRNA described herein can beused to express a ligand or ligand receptor on the surface of a cell(e.g., a homing moiety). A ligand or ligand receptor moiety attached toa cell surface can permit the cell to have a desired biologicalinteraction with a tissue or an agent in vivo. A ligand can be anantibody, an antibody fragment, an aptamer, a peptide, a vitamin, acarbohydrate, a protein or polypeptide, a receptor, e.g., cell-surfacereceptor, an adhesion molecule, a glycoprotein, a sugar residue, atherapeutic agent, a drug, a glycosaminoglycan, or any combinationthereof. For example, a ligand can be an antibody that recognizes acancer-cell specific antigen, rendering the cell capable ofpreferentially interacting with tumor cells to permit tumor-specificlocalization of a modified cell. A ligand can confer the ability of acell composition to accumulate in a tissue to be treated, since apreferred ligand may be capable of interacting with a target molecule onthe external face of a tissue to be treated. Ligands having limitedcross-reactivity to other tissues are generally preferred.

In some cases, a ligand can act as a homing moiety which permits thecell to target to a specific tissue or interact with a specific ligand.Such homing moieties can include, but are not limited to, any member ofa specific binding pair, antibodies, monoclonal antibodies, orderivatives or analogs thereof, including without limitation: Fvfragments, single chain Fv (scFv) fragments, Fab′ fragments, F(ab′)2fragments, single domain antibodies, camelized antibodies and antibodyfragments, humanized antibodies and antibody fragments, and multivalentversions of the foregoing; multivalent binding reagents includingwithout limitation: monospecific or bispecific antibodies, such asdisulfide stabilized Fv fragments, scFv tandems ((SCFV)₂ fragments),diabodies, tribodies or tetrabodies, which typically are covalentlylinked or otherwise stabilized (i.e., leucine zipper or helixstabilized) scFv fragments; and other homing moieties include forexample, aptamers, receptors, and fusion proteins.

In some embodiments, the homing moiety may be a surface-bound antibody,which can permit tuning of cell targeting specificity. This isespecially useful since highly specific antibodies can be raised againstan epitope of interest for the desired targeting site. In oneembodiment, multiple antibodies are expressed on the surface of a cell,and each antibody can have a different specificity for a desired target.Such approaches can increase the avidity and specificity of hominginteractions.

A skilled artisan can select any homing moiety based on the desiredlocalization or function of the cell, for example an estrogen receptorligand, such as tamoxifen, can target cells to estrogen-dependent breastcancer cells that have an increased number of estrogen receptors on thecell surface. Other non-limiting examples of ligand/receptorinteractions include CCR1 (e.g., for treatment of inflamed joint tissuesor brain in rheumatoid arthritis, and/or multiple sclerosis), CCR7, CCR8(e.g., targeting to lymph node tissue), CCR6, CCR9,CCR10 (e.g., totarget to intestinal tissue), CCR4, CCR10 (e.g., for targeting to skin),CXCR4 (e.g., for general enhanced transmigration), HCELL (e.g., fortreatment of inflammation and inflammatory disorders, bone marrow),Alpha4beta7 (e.g., for intestinal mucosa targeting), VLA-4NCAM-1 (e.g.,targeting to endothelium). In general, any receptor involved intargeting (e.g., cancer metastasis) can be harnessed for use in themethods and compositions described herein.

Modulation of Cell Lineage

Provided are methods of inducing an alteration in cell fate in a targetmammalian cell. The target mammalian cell may be a precursor cell andthe alteration may involve driving differentiation into a lineage, orblocking such differentiation. Alternatively, the target mammalian cellmay be a differentiated cell, and the cell fate alteration includesdriving de-differentiation into a pluripotent precursor cell, orblocking such de-differentiation, such as the dedifferentiation ofcancer cells into cancer stem cells. In situations where a change incell fate is desired, effective amounts of mRNAs encoding a cell fateinductive polypeptide is introduced into a target cell under conditionssuch that an alteration in cell fate is induced. In some embodiments,the modified mRNAs are useful to reprogram a subpopulation of cells froma first phenotype to a second phenotype. Such a reprogramming may betemporary or permanent.

Optionally, the reprogramming induces a target cell to adopt anintermediate phenotype.

Additionally, the methods of the present invention are particularlyuseful to generate induced pluripotent stem cells (iPS cells) because ofthe high efficiency of transfection, the ability to re-transfect cells,and the tenability of the amount of recombinant polypeptides produced inthe target cells. Further, the use of iPS cells generated using themethods described herein is expected to have a reduced incidence ofteratoma formation.

Also provided are methods of reducing cellular differentiation in atarget cell population. For example, a target cell population containingone or more precursor cell types is contacted with a composition havingan effective amount of a polynucleotides, primary constructs and mmRNAencoding a polypeptide, under conditions such that the polypeptide istranslated and reduces the differentiation of the precursor cell. Innon-limiting embodiments, the target cell population contains injuredtissue in a mammalian subject or tissue affected by a surgicalprocedure. The precursor cell is, e.g., a stromal precursor cell, aneural precursor cell, or a mesenchymal precursor cell.

In a specific embodiment, provided are polynucleotide, primary constructor mmRNA that encode one or more differentiation factors Gata4, Mef2cand Tbx4. These mRNA-generated factors are introduced into fibroblastsand drive the reprogramming into cardiomyocytes. Such a reprogrammingcan be performed in vivo, by contacting an mRNA-containing patch orother material to damaged cardiac tissue to facilitate cardiacregeneration. Such a process promotes cardiomyocyte genesis as opposedto fibrosis.

Mediation of Cell Death

In one embodiment, polynucleotides, primary constructs or mmRNAcompositions can be used to induce apoptosis in a cell (e.g., a cancercell) by increasing the expression of a death receptor, a death receptorligand or a combination thereof. This method can be used to induce celldeath in any desired cell and has particular usefulness in the treatmentof cancer where cells escape natural apoptotic signals.

Apoptosis can be induced by multiple independent signaling pathways thatconverge upon a final effector mechanism consisting of multipleinteractions between several “death receptors” and their ligands, whichbelong to the tumor necrosis factor (TNF) receptor/ligand superfamily.The best-characterized death receptors are CD95 (“Fas”), TNFR1 (p55),death receptor 3 (DR3 or Apo3/TRAMO), DR4 and DR5 (apo2-TRAIL-R2). Thefinal effector mechanism of apoptosis may be the activation of a seriesof proteinases designated as caspases. The activation of these caspasesresults in the cleavage of a series of vital cellular proteins and celldeath. The molecular mechanism of death receptors/ligands-inducedapoptosis is well known in the art. For example, Fas/FasL-mediatedapoptosis is induced by binding of three FasL molecules which inducestrimerization of Fas receptor via C-terminus death domains (DDs), whichin turn recruits an adapter protein FADD (Fas-associated protein withdeath domain) and Caspase-8. The oligomerization of this trimolecularcomplex, Fas/FAIDD/caspase-8, results in proteolytic cleavage ofproenzyme caspase-8 into active caspase-8 that, in turn, initiates theapoptosis process by activating other downstream caspases throughproteolysis, including caspase-3. Death ligands in general are apoptoticwhen formed into trimers or higher order of structures. As monomers,they may serve as antiapoptotic agents by competing with the trimers forbinding to the death receptors.

In one embodiment, the polynucleotides, primary constructs or mmRNAcomposition encodes for a death receptor (e.g., Fas, TRAIL, TRAMO, TNFR,TLR etc). Cells made to express a death receptor by transfection ofpolynucleotides, primary constructs and mmRNA become susceptible todeath induced by the ligand that activates that receptor. Similarly,cells made to express a death ligand, e.g., on their surface, willinduce death of cells with the receptor when the transfected cellcontacts the target cell.

In another embodiment, the polynucleotides, primary constructs and mmRNAcomposition encodes for a death receptor ligand (e.g., FasL, TNF, etc).In another embodiment, the polynucleotides, primary constructs and mmRNAcomposition encodes a caspase (e.g., caspase 3, caspase 8, caspase 9etc). Where cancer cells often exhibit a failure to properlydifferentiate to a non-proliferative or controlled proliferative form,in another embodiment, the synthetic, polynucleotides, primaryconstructs and mmRNA composition encodes for both a death receptor andits appropriate activating ligand. In another embodiment, the synthetic,polynucleotides, primary constructs and mmRNA composition encodes for adifferentiation factor that when expressed in the cancer cell, such as acancer stem cell, will induce the cell to differentiate to anon-pathogenic or nonself-renewing phenotype (e.g., reduced cell growthrate, reduced cell division etc) or to induce the cell to enter adormant cell phase (e.g., G₀ resting phase).

One of skill in the art will appreciate that the use ofapoptosis-inducing techniques may require that the polynucleotides,primary constructs or mmRNA are appropriately targeted to e.g., tumorcells to prevent unwanted wide-spread cell death. Thus, one can use adelivery mechanism (e.g., attached ligand or antibody, targeted liposomeetc) that recognizes a cancer antigen such that the polynucleotides,primary constructs or mmRNA are expressed only in cancer cells.

Cosmetic Applications

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be used in the treatment, amelioration or prophylaxis of cosmeticconditions. Such conditions include acne, rosacea, scarring, wrinkles,eczema, shingles, psoriasis, age spots, birth marks, dry skin, calluses,rash (e.g., diaper, heat), scabies, hives, warts, insect bites,vitiligo, dandruff, freckles, and general signs of aging.

VI. Kits and Devices Kits

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods of the present invention. Typicallykits will comprise sufficient amounts and/or numbers of components toallow a user to perform multiple treatments of a subject(s) and/or toperform multiple experiments.

In one aspect, the present invention provides kits comprising themolecules (polynucleotides, primary constructs or mmRNA) of theinvention. In one embodiment, the kit comprises one or more functionalantibodies or function fragments thereof.

Said kits can be for protein production, comprising a firstpolynucleotide, primary construct or mmRNA comprising a translatableregion. The kit may further comprise packaging and instructions and/or adelivery agent to form a formulation composition. The delivery agent maycomprise a saline, a buffered solution, a lipidoid or any delivery agentdisclosed herein.

In one embodiment, the buffer solution may include sodium chloride,calcium chloride, phosphate and/or EDTA. In another embodiment, thebuffer solution may include, but is not limited to, saline, saline with2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5%Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodiumchloride with 2 mM calcium and mannose (See e.g., U.S. Pub. No.20120258046; herein incorporated by reference in its entirety). In afurther embodiment, the buffer solutions may be precipitated or it maybe lyophilized. The amount of each component may be varied to enableconsistent, reproducible higher concentration saline or simple bufferformulations. The components may also be varied in order to increase thestability of modified RNA in the buffer solution over a period of timeand/or under a variety of conditions. In one aspect, the presentinvention provides kits for protein production, comprising: apolynucleotide, primary construct or mmRNA comprising a translatableregion, provided in an amount effective to produce a desired amount of aprotein encoded by the translatable region when introduced into a targetcell; a second polynucleotide comprising an inhibitory nucleic acid,provided in an amount effective to substantially inhibit the innateimmune response of the cell; and packaging and instructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a polynucleotide, primary construct or mmRNAcomprising a translatable region, wherein the polynucleotide exhibitsreduced degradation by a cellular nuclease, and packaging andinstructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a polynucleotide, primary construct or mmRNAcomprising a translatable region, wherein the polynucleotide exhibitsreduced degradation by a cellular nuclease, and a mammalian cellsuitable for translation of the translatable region of the first nucleicacid.

In one embodiment, the levels of Protein C may be measured byimmunoassay. The assay may be purchased and is available from any numberof suppliers including BioMerieux, Inc. (Durham, N.C.), AbbottLaboratories (Abbott Park, Ill.), Siemens Medical Solutions USA, Inc.(Malvern, Pa.), BIOPORTO® Diagnostics A/S (Gentofte, Denmark), USCN®Life Science Inc. (Houston, Tex.) or Roche Diagnostic Corporation(Indianapolis, Ind.). In this embodiment, the assay may be used toassess levels of Protein C or its activated form or a variant deliveredas or in response to administration of a modified mRNA molecule.

Devices

The present invention provides for devices which may incorporatepolynucleotides, primary constructs or mmRNA that encode polypeptides ofinterest. These devices contain in a stable formulation the reagents tosynthesize a polynucleotide in a formulation available to be immediatelydelivered to a subject in need thereof, such as a human patient.Non-limiting examples of such a polypeptide of interest include a growthfactor and/or angiogenesis stimulator for wound healing, a peptideantibiotic to facilitate infection control, and an antigen to rapidlystimulate an immune response to a newly identified virus.

Devices may also be used in conjunction with the present invention. Inone embodiment, a device is used to assess levels of a protein which hasbeen administered in the form of a modified mRNA. The device maycomprise a blood, urine or other biofluidic test. It may be as large asto include an automated central lab platform or a small decentralizedbench top device. It may be point of care or a handheld device. In thisembodiment, for example, Protein C or APC may be quatitated before,during or after treatment with a modified mRNA encoding Protein C (itszymogen), APC or any variants thereof. Protein C, also known asautoprothrombin HA and blood coagulation factor XIV is a zymogen, orprecursor, of a serine protease which plays an important role in theregulation of blood coagulation and generation of fibrinolytic activityin vivo. It is synthesized in the liver as a single-chain polypeptidebut undergoes posttranslational processing to give rise to a two-chainintermediate. The intermediate form of Protein C is converted viathrombin-mediated cleavage of a 12-residue peptide from theamino-terminus of the heavy chain to of the molecule to the active form,known as “activated protein C” (APC). The device may be useful in drugdiscovery efforts as a companion diagnostic test associated with ProteinC, or APC treatment such as for sepsis or severe sepsis. In earlystudies it was suggested that APC had the ability to reduce mortality insevere sepsis. Following this line of work, clinical studies lead to theFDA approval of one compound, activated drotrecogin alfa (recombinantprotein C). However, in late 2011, the drug was withdrawn from sale inall markets following results of the PROWESS-SHOCK study, which showedthe study did not meet the primary endpoint of a statisticallysignificant reduction in 28-day all-cause mortality in patients withseptic shock. The present invention provides modified mRNA moleculeswhich may be used in the diagnosis and treatment of sepsis, severesepsis and septicemia which overcome prior issues or problems associatedwith increasing protein expression efficiencies in mammals.

In some embodiments the device is self-contained, and is optionallycapable of wireless remote access to obtain instructions for synthesisand/or analysis of the generated polynucleotide, primary construct ormmRNA. The device is capable of mobile synthesis of at least onepolynucleotide, primary construct or mmRNA and preferably an unlimitednumber of different polynucleotides, primary constructs or mmRNA. Incertain embodiments, the device is capable of being transported by oneor a small number of individuals. In other embodiments, the device isscaled to fit on a benchtop or desk. In other embodiments, the device isscaled to fit into a suitcase, backpack or similarly sized object. Inanother embodiment, the device may be a point of care or handhelddevice. In further embodiments, the device is scaled to fit into avehicle, such as a car, truck or ambulance, or a military vehicle suchas a tank or personnel carrier. The information necessary to generate amodified mRNA encoding polypeptide of interest is present within acomputer readable medium present in the device.

In one embodiment, a device may be used to assess levels of a proteinwhich has been administered in the form of a polynucleotide, primaryconstruct or mmRNA. The device may comprise a blood, urine or otherbiofluidic test.

In some embodiments, the device is capable of communication (e.g.,wireless communication) with a database of nucleic acid and polypeptidesequences. The device contains at least one sample block for insertionof one or more sample vessels. Such sample vessels are capable ofaccepting in liquid or other form any number of materials such astemplate DNA, nucleotides, enzymes, buffers, and other reagents. Thesample vessels are also capable of being heated and cooled by contactwith the sample block. The sample block is generally in communicationwith a device base with one or more electronic control units for the atleast one sample block. The sample block preferably contains a heatingmodule, such heating molecule capable of heating and/or cooling thesample vessels and contents thereof to temperatures between about −20Cand above +100C. The device base is in communication with a voltagesupply such as a battery or external voltage supply. The device alsocontains means for storing and distributing the materials for RNAsynthesis.

Optionally, the sample block contains a module for separating thesynthesized nucleic acids. Alternatively, the device contains aseparation module operably linked to the sample block. Preferably thedevice contains a means for analysis of the synthesized nucleic acid.Such analysis includes sequence identity (demonstrated such as byhybridization), absence of non-desired sequences, measurement ofintegrity of synthesized mRNA (such has by microfluidic viscometrycombined with spectrophotometry), and concentration and/or potency ofmodified RNA (such as by spectrophotometry).

In certain embodiments, the device is combined with a means fordetection of pathogens present in a biological material obtained from asubject, e.g., the IBIS PLEX-ID system (Abbott, Abbott Park, Ill.) formicrobial identification.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662; each of which is hereinincorporated by reference in their entirety. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 (herein incorporated by reference in itsentirety) and functional equivalents thereof. Jet injection deviceswhich deliver liquid compositions to the dermis via a liquid jetinjector and/or via a needle which pierces the stratum corneum andproduces a jet which reaches the dermis are suitable. Jet injectiondevices are described, for example, in U.S. Pat. Nos. 5,480,381;5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911;5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627;5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; andPCT publications WO 97/37705 and WO 97/13537; each of which are hreinincorporated by reference in their entirety. Ballistic powder/particledelivery devices which use compressed gas to accelerate vaccine inpowder form through the outer layers of the skin to the dermis aresuitable. Alternatively or additionally, conventional syringes may beused in the classical mantoux method of intradermal administration.

In some embodiments, the device may be a pump or comprise a catheter foradministration of compounds or compositions of the invention across theblood brain barrier. Such devices include but are not limited to apressurized olfactory delivery device, iontophoresis devices,multi-layered microfluidic devices, and the like. Such devices may beportable or stationary. They may be implantable or externally tetheredto the body or combinations thereof.

Devices for administration may be employed to deliver thepolynucleotides, primary constructs or mmRNA of the present inventionaccording to single, multi- or split-dosing regimens taught herein. Suchdevices are described below.

Method and devices known in the art for multi-administration to cells,organs and tissues are contemplated for use in conjunction with themethods and compositions disclosed herein as embodiments of the presentinvention. These include, for example, those methods and devices havingmultiple needles, hybrid devices employing for example lumens orcatheters as well as devices utilizing heat, electric current orradiation driven mechanisms.

According to the present invention, these multi-administration devicesmay be utilized to deliver the single, multi- or split dosescontemplated herein.

A method for delivering therapeutic agents to a solid tissue has beendescribed by Bahrami et al. and is taught for example in US PatentPublication 20110230839, the contents of which are incorporated hereinby reference in their entirety. According to Bahrami, an array ofneedles is incorporated into a device which delivers a substantiallyequal amount of fluid at any location in said solid tissue along eachneedle's length.

A device for delivery of biological material across the biologicaltissue has been described by Kodgule et al. and is taught for example inUS Patent Publication 20110172610, the contents of which areincorporated herein by reference in their entirety. According toKodgule, multiple hollow micro-needles made of one or more metals andhaving outer diameters from about 200 microns to about 350 microns andlengths of at least 100 microns are incorporated into the device whichdelivers peptides, proteins, carbohydrates, nucleic acid molecules,lipids and other pharmaceutically active ingredients or combinationsthereof.

A delivery probe for delivering a therapeutic agent to a tissue has beendescribed by Gunday et al. and is taught for example in US PatentPublication 20110270184, the contents of each of which are incorporatedherein by reference in their entirety. According to Gunday, multipleneedles are incorporated into the device which moves the attachedcapsules between an activated position and an inactivated position toforce the agent out of the capsules through the needles.

A multiple-injection medical apparatus has been described by Assaf andis taught for example in US Patent Publication 20110218497, the contentsof which are incorporated herein by reference in their entirety.According to Assaf, multiple needles are incorporated into the devicewhich has a chamber connected to one or more of said needles and a meansfor continuously refilling the chamber with the medical fluid after eachinjection.

In one embodiment, the polynucleotide, primary construct, or mmRNA isadministered subcutaneously or intramuscularly via at least 3 needles tothree different, optionally adjacent, sites simultaneously, or within a60 minutes period (e.g., administration to 4, 5, 6, 7, 8, 9, or 10 sitessimultaneously or within a 60 minute period). The split doses can beadministered simultaneously to adjacent tissue using the devicesdescribed in U.S. Patent Publication Nos. 20110230839 and 20110218497,each of which is incorporated herein by reference in their entirety.

An at least partially implantable system for injecting a substance intoa patient's body, in particular a penis erection stimulation system hasbeen described by Forsell and is taught for example in US PatentPublication 20110196198, the contents of which are incorporated hereinby reference in their entirety. According to Forsell, multiple needlesare incorporated into the device which is implanted along with one ormore housings adjacent the patient's left and right corpora cavernosa. Areservoir and a pump are also implanted to supply drugs through theneedles.

A method for the transdermal delivery of a therapeutic effective amountof iron has been described by Berenson and is taught for example in USPatent Publication 20100130910, the contents of which are incorporatedherein by reference in their entirety. According to Berenson, multipleneedles may be used to create multiple micro channels in stratum corneumto enhance transdermal delivery of the ionic iron on an iontophoreticpatch.

A method for delivery of biological material across the biologicaltissue has been described by Kodgule et al and is taught for example inUS Patent Publication 20110196308, the contents of which areincorporated herein by reference in their entirety. According toKodgule, multiple biodegradable microneedles containing a therapeuticactive ingredient are incorporated in a device which delivers proteins,carbohydrates, nucleic acid molecules, lipids and other pharmaceuticallyactive ingredients or combinations thereof.

A transdermal patch comprising a botulinum toxin composition has beendescribed by Donovan and is taught for example in US Patent Publication20080220020, the contents of which are incorporated herein by referencein their entirety. According to Donovan, multiple needles areincorporated into the patch which delivers botulinum toxin under stratumcorneum through said needles which project through the stratum corneumof the skin without rupturing a blood vessel.

A small, disposable drug reservoir, or patch pump, which can holdapproximately 0.2 to 15 mL of liquid formulations can be placed on theskin and deliver the formulation continuously subcutaneously using asmall bore needed (e.g., 26 to 34 gauge). As non-limiting examples, thepatch pump may be 50 mm by 76 mm by 20 mm spring loaded having a 30 to34 gauge needle (BD™ Microinfuser, Franklin Lakes N.J.), 41 mm by 62 mmby 17 mm with a 2 mL reservoir used for drug delivery such as insulin(OMNIPOD®, Insulet Corporation Bedford, Mass.), or 43-60 mm diameter, 10mm thick with a 0.5 to 10 mL reservoir (PATCHPUMP®, SteadyMedTherapeutics, San Francisco, Calif.). Further, the patch pump may bebattery powered and/or rechargeable.

A cryoprobe for administration of an active agent to a location ofcryogenic treatment has been described by Toubia and is taught forexample in US Patent Publication 20080140061, the contents of which areincorporated herein by reference in their entirety. According to Toubia,multiple needles are incorporated into the probe which receives theactive agent into a chamber and administers the agent to the tissue.

A method for treating or preventing inflammation or promoting healthyjoints has been described by Stock et al and is taught for example in USPatent Publication 20090155186, the contents of which are incorporatedherein by reference in their entirety. According to Stock, multipleneedles are incorporated in a device which administers compositionscontaining signal transduction modulator compounds.

A multi-site injection system has been described by Kimmell et al. andis taught for example in US Patent Publication 20100256594, the contentsof which are incorporated herein by reference in their entirety.According to Kimmell, multiple needles are incorporated into a devicewhich delivers a medication into a stratum corneum through the needles.

A method for delivering interferons to the intradermal compartment hasbeen described by Dekker et al. and is taught for example in US PatentPublication 20050181033, the contents of which are incorporated hereinby reference in their entirety. According to Dekker, multiple needleshaving an outlet with an exposed height between 0 and 1 mm areincorporated into a device which improves pharmacokinetics andbioavailability by delivering the substance at a depth between 0.3 mmand 2 mm.

A method for delivering genes, enzymes and biological agents to tissuecells has described by Desai and is taught for example in US PatentPublication 20030073908, the contents of which are incorporated hereinby reference in their entirety. According to Desai, multiple needles areincorporated into a device which is inserted into a body and delivers amedication fluid through said needles.

A method for treating cardiac arrhythmias with fibroblast cells has beendescribed by Lee et al and is taught for example in US PatentPublication 20040005295, the contents of which are incorporated hereinby reference in their entirety. According to Lee, multiple needles areincorporated into the device which delivers fibroblast cells into thelocal region of the tissue.

A method using a magnetically controlled pump for treating a brain tumorhas been described by Shachar et al. and is taught for example in U.S.Pat. No. 7,799,012 (method) and 7799016 (device), the contents of whichare incorporated herein by reference in their entirety. AccordingShachar, multiple needles were incorporated into the pump which pushes amedicating agent through the needles at a controlled rate.

Methods of treating functional disorders of the bladder in mammalianfemales have been described by Versi et al. and are taught for examplein U.S. Pat. No. 8,029,496, the contents of which are incorporatedherein by reference in their entirety. According to Versi, an array ofmicro-needles is incorporated into a device which delivers a therapeuticagent through the needles directly into the trigone of the bladder.

A micro-needle transdermal transport device has been described by Angelet al and is taught for example in U.S. Pat. No. 7,364,568, the contentsof which are incorporated herein by reference in their entirety.According to Angel, multiple needles are incorporated into the devicewhich transports a substance into a body surface through the needleswhich are inserted into the surface from different directions. Themicro-needle transdermal transport device may be a solid micro-needlesystem or a hollow micro-needle system. As a non-limiting example, thesolid micro-needle system may have up to a 0.5 mg capacity, with300-1500 solid micro-needles per cm² about 150-700 lam tall coated witha drug. The micro-needles penetrate the stratum corneum and remain inthe skin for short duration (e.g., 20 seconds to 15 minutes). In anotherexample, the hollow micro-needle system has up to a 3 mL capacity todeliver liquid formulations using 15-20 microneedles per cm2 beingapproximately 950 μm tall. The micro-needles penetrate the skin to allowthe liquid formulations to flow from the device into the skin. Thehollow micro-needle system may be worn from 1 to 30 minutes depending onthe formulation volume and viscocity.

A device for subcutaneous infusion has been described by Dalton et aland is taught for example in U.S. Pat. No. 7,150,726, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Dalton, multiple needles are incorporated into the device whichdelivers fluid through the needles into a subcutaneous tissue.

A device and a method for intradermal delivery of vaccines and genetherapeutic agents through microcannula have been described by Miksztaet al. and are taught for example in U.S. Pat. No. 7,473,247, thecontents of which are incorporated herein by reference in theirentirety. According to Mitszta, at least one hollow micro-needle isincorporated into the device which delivers the vaccines to thesubject's skin to a depth of between 0.025 mm and 2 mm.

A method of delivering insulin has been described by Pettis et al and istaught for example in U.S. Pat. No. 7,722,595, the contents of which areincorporated herein by reference in their entirety. According to Pettis,two needles are incorporated into a device wherein both needles insertessentially simultaneously into the skin with the first at a depth ofless than 2.5 mm to deliver insulin to intradermal compartment and thesecond at a depth of greater than 2.5 mm and less than 5.0 mm to deliverinsulin to subcutaneous compartment.

Cutaneous injection delivery under suction has been described byKochamba et al. and is taught for example in U.S. Pat. No. 6,896,666,the contents of which are incorporated herein by reference in theirentirety. According to Kochamba, multiple needles in relative adjacencywith each other are incorporated into a device which injects a fluidbelow the cutaneous layer.

A device for withdrawing or delivering a substance through the skin hasbeen described by Down et al and is taught for example in U.S. Pat. No.6,607,513, the contents of which are incorporated herein by reference intheir entirety. According to Down, multiple skin penetrating memberswhich are incorporated into the device have lengths of about 100 micronsto about 2000 microns and are about 30 to 50 gauge.

A device for delivering a substance to the skin has been described byPalmer et al and is taught for example in U.S. Pat. No. 6,537,242, thecontents of which are incorporated herein by reference in theirentirety. According to Palmer, an array of micro-needles is incorporatedinto the device which uses a stretching assembly to enhance the contactof the needles with the skin and provides a more uniform delivery of thesubstance.

A perfusion device for localized drug delivery has been described byZamoyski and is taught for example in U.S. Pat. No. 6,468,247, thecontents of which are incorporated herein by reference in theirentirety. According to Zamoyski, multiple hypodermic needles areincorporated into the device which injects the contents of thehypodermics into a tissue as said hypodermics are being retracted.

A method for enhanced transport of drugs and biological molecules acrosstissue by improving the interaction between micro-needles and human skinhas been described by Prausnitz et al. and is taught for example in U.S.Pat. No. 6,743,211, the contents of which are incorporated herein byreference in their entirety. According to Prausnitz, multiplemicro-needles are incorporated into a device which is able to present amore rigid and less deformable surface to which the micro-needles areapplied.

A device for intraorgan administration of medicinal agents has beendescribed by Ting et al and is taught for example in U.S. Pat. No.6,077,251, the contents of which are incorporated herein by reference intheir entirety. According to Ting, multiple needles having side openingsfor enhanced administration are incorporated into a device which byextending and retracting said needles from and into the needle chamberforces a medicinal agent from a reservoir into said needles and injectssaid medicinal agent into a target organ.

A multiple needle holder and a subcutaneous multiple channel infusionport has been described by Brown and is taught for example in U.S. Pat.No. 4,695,273, the contents of which are incorporated herein byreference in their entirety. According to Brown, multiple needles on theneedle holder are inserted through the septum of the infusion port andcommunicate with isolated chambers in said infusion port.

A dual hypodermic syringe has been described by Horn and is taught forexample in U.S. Pat. No. 3,552,394, the contents of which areincorporated herein by reference in their entirety. According to Horn,two needles incorporated into the device are spaced apart less than 68mm and may be of different styles and lengths, thus enabling injectionsto be made to different depths.

A syringe with multiple needles and multiple fluid compartments has beendescribed by Hershberg and is taught for example in U.S. Pat. No.3,572,336, the contents of which are incorporated herein by reference intheir entirety. According to Hershberg, multiple needles areincorporated into the syringe which has multiple fluid compartments andis capable of simultaneously administering incompatible drugs which arenot able to be mixed for one injection.

A surgical instrument for intradermal injection of fluids has beendescribed by Eliscu et al. and is taught for example in U.S. Pat. No.2,588,623, the contents of which are incorporated herein by reference intheir entirety. According to Eliscu, multiple needles are incorporatedinto the instrument which injects fluids intradermally with a widerdisperse.

An apparatus for simultaneous delivery of a substance to multiple breastmilk ducts has been described by Hung and is taught for example in EP1818017, the contents of which are incorporated herein by reference intheir entirety. According to Hung, multiple lumens are incorporated intothe device which inserts though the orifices of the ductal networks anddelivers a fluid to the ductal networks.

A catheter for introduction of medications to the tissue of a heart orother organs has been described by Tkebuchava and is taught for examplein WO2006138109, the contents of which are incorporated herein byreference in their entirety. According to Tkebuchava, two curved needlesare incorporated which enter the organ wall in a flattened trajectory.

Devices for delivering medical agents have been described by Mckay etal. and are taught for example in WO2006118804, the content of which areincorporated herein by reference in their entirety. According to Mckay,multiple needles with multiple orifices on each needle are incorporatedinto the devices to facilitate regional delivery to a tissue, such asthe interior disc space of a spinal disc.

A method for directly delivering an immunomodulatory substance into anintradermal space within a mammalian skin has been described by Pettisand is taught for example in WO2004020014, the contents of which areincorporated herein by reference in their entirety. According to Pettis,multiple needles are incorporated into a device which delivers thesubstance through the needles to a depth between 0.3 mm and 2 mm.

Methods and devices for administration of substances into at least twocompartments in skin for systemic absorption and improvedpharmacokinetics have been described by Pettis et al. and are taught forexample in WO2003094995, the contents of which are incorporated hereinby reference in their entirety. According to Pettis, multiple needleshaving lengths between about 300 μm and about 5 mm are incorporated intoa device which delivers to intradermal and subcutaneous tissuecompartments simultaneously.

A drug delivery device with needles and a roller has been described byZimmerman et al. and is taught for example in WO2012006259, the contentsof which are incorporated herein by reference in their entirety.According to Zimmerman, multiple hollow needles positioned in a rollerare incorporated into the device which delivers the content in areservoir through the needles as the roller rotates.

A drug delivery device such as a stent is known in the art and is taughtfor example in U.S. Pat. No. 8,333,799, U.S. Pub. Nos. US20060020329,US20040172127 and US20100161032; the contents of each of which areherein incorporated by reference in their entirety. Formulations of thepolynucleotides, primary constructs, mmRNA described herein may bedelivered using stents. Additionally, stents used herein may be able todeliver multiple polynucleotides, primary constructs and/or mmRNA and/orformulations at the same or varied rates of delivery. Non-limitingexamples of manufacturers of stents include CORDIS® (Miami, Fla.)(CYPHER®), Boston Scientific Corporation (Natick, Mass.) (TAXUS®),Medtronic (Minneapolis, Minn.) (ENDEAVOUR®) and Abbott (Abbott Park,Ill.) (XIENCE V®).

Methods and Devices Utilizing Catheters and/or Lumens

Methods and devices using catheters and lumens may be employed toadminister the mmRNA of the present invention on a single, multi- orsplit dosing schedule. Such methods and devices are described below.

A catheter-based delivery of skeletal myoblasts to the myocardium ofdamaged hearts has been described by Jacoby et al and is taught forexample in US Patent Publication 20060263338, the contents of which areincorporated herein by reference in their entirety. According to Jacoby,multiple needles are incorporated into the device at least part of whichis inserted into a blood vessel and delivers the cell compositionthrough the needles into the localized region of the subject's heart.

An apparatus for treating asthma using neurotoxin has been described byDeem et al and is taught for example in US Patent Publication20060225742, the contents of which are incorporated herein by referencein their entirety. According to Deem, multiple needles are incorporatedinto the device which delivers neurotoxin through the needles into thebronchial tissue.

A method for administering multiple-component therapies has beendescribed by Nayak and is taught for example in U.S. Pat. No. 7,699,803,the contents of which are incorporated herein by reference in theirentirety. According to Nayak, multiple injection cannulas may beincorporated into a device wherein depth slots may be included forcontrolling the depth at which the therapeutic substance is deliveredwithin the tissue.

A surgical device for ablating a channel and delivering at least onetherapeutic agent into a desired region of the tissue has been describedby McIntyre et al and is taught for example in U.S. Pat. No. 8,012,096,the contents of which are incorporated herein by reference in theirentirety. According to McIntyre, multiple needles are incorporated intothe device which dispenses a therapeutic agent into a region of tissuesurrounding the channel and is particularly well suited fortransmyocardial revascularization operations.

Methods of treating functional disorders of the bladder in mammalianfemales have been described by Versi et al and are taught for example inU.S. Pat. No. 8,029,496, the contents of which are incorporated hereinby reference in their entirety. According to Versi, an array ofmicro-needles is incorporated into a device which delivers a therapeuticagent through the needles directly into the trigone of the bladder.

A device and a method for delivering fluid into a flexible biologicalbarrier have been described by Yeshurun et al. and are taught forexample in U.S. Pat. No. 7,998,119 (device) and 8,007,466 (method), thecontents of which are incorporated herein by reference in theirentirety. According to Yeshurun, the micro-needles on the devicepenetrate and extend into the flexible biological barrier and fluid isinjected through the bore of the hollow micro-needles.

A method for epicardially injecting a substance into an area of tissueof a heart having an epicardial surface and disposed within a torso hasbeen described by Bonner et al and is taught for example in U.S. Pat.No. 7,628,780, the contents of which are incorporated herein byreference in their entirety. According to Bonner, the devices haveelongate shafts and distal injection heads for driving needles intotissue and injecting medical agents into the tissue through the needles.

A device for sealing a puncture has been described by Nielsen et al andis taught for example in U.S. Pat. No. 7,972,358, the contents of whichare incorporated herein by reference in their entirety. According toNielsen, multiple needles are incorporated into the device whichdelivers a closure agent into the tissue surrounding the puncture tract.

A method for myogenesis and angiogenesis has been described by Chiu etal. and is taught for example in U.S. Pat. No. 6,551,338, the contentsof which are incorporated herein by reference in their entirety.According to Chiu, 5 to 15 needles having a maximum diameter of at least1.25 mm and a length effective to provide a puncture depth of 6 to 20 mmare incorporated into a device which inserts into proximity with amyocardium and supplies an exogeneous angiogenic or myogenic factor tosaid myocardium through the conduits which are in at least some of saidneedles.

A method for the treatment of prostate tissue has been described byBolmsj et al. and is taught for example in U.S. Pat. No. 6,524,270, thecontents of which are incorporated herein by reference in theirentirety. According to Bolmsj, a device comprising a catheter which isinserted through the urethra has at least one hollow tip extendible intothe surrounding prostate tissue. An astringent and analgesic medicine isadministered through said tip into said prostate tissue.

A method for infusing fluids to an intraosseous site has been describedby Findlay et al. and is taught for example in U.S. Pat. No. 6,761,726,the contents of which are incorporated herein by reference in theirentirety. According to Findlay, multiple needles are incorporated into adevice which is capable of penetrating a hard shell of material coveredby a layer of soft material and delivers a fluid at a predetermineddistance below said hard shell of material.

A device for injecting medications into a vessel wall has been describedby Vigil et al. and is taught for example in U.S. Pat. No. 5,713,863,the contents of which are incorporated herein by reference in theirentirety. According to Vigil, multiple injectors are mounted on each ofthe flexible tubes in the device which introduces a medication fluidthrough a multi-lumen catheter, into said flexible tubes and out of saidinjectors for infusion into the vessel wall.

A catheter for delivering therapeutic and/or diagnostic agents to thetissue surrounding a bodily passageway has been described by Faxon etal. and is taught for example in U.S. Pat. No. 5,464,395, the contentsof which are incorporated herein by reference in their entirety.According to Faxon, at least one needle cannula is incorporated into thecatheter which delivers the desired agents to the tissue through saidneedles which project outboard of the catheter.

Balloon catheters for delivering therapeutic agents have been describedby Orr and are taught for example in WO2010024871, the contents of whichare incorporated herein by reference in their entirety. According toOrr, multiple needles are incorporated into the devices which deliverthe therapeutic agents to different depths within the tissue. In anotheraspect, drug-eluting balloons may be used to deliver the formulationsdescribed herein. The drug-eluting balloons may be used in target lesionapplications such as, but are not limited to, in-stent restenosis,treating lesion in tortuous vessels, bifurcation lesions,femoral/popliteal lesions and below the knee lesions.

A device for deliverying therapeutic agents (e.g., polynucleotides,primary constructs or mmRNA) to tissue disposed about a lumin has beendescribed by Perry et al. and is taught for example in U.S. Pat. Pub.US20100125239, the contents of which are herein incorporated byreference in their entirety. According to Perry, the catheter has aballoon which may be coated with a therapeutic agent by methods known inthe art and described in Perry. When the balloon expands, thetherapeutic agent will contact the surrounding tissue. The device mayadditionally have a heat source to change the temperature of the coatingon the balloon to release the thereapeutic agent to the tissue.

Methods and Devices Utilizing Electrical Current

Methods and devices utilizing electric current may be employed todeliver the mmRNA of the present invention according to the single,multi- or split dosing regimens taught herein. Such methods and devicesare described below.

An electro collagen induction therapy device has been described byMarquez and is taught for example in US Patent Publication 20090137945,the contents of which are incorporated herein by reference in theirentirety. According to Marquez, multiple needles are incorporated intothe device which repeatedly pierce the skin and draw in the skin aportion of the substance which is applied to the skin first.

An electrokinetic system has been described by Etheredge et al. and istaught for example in US Patent Publication 20070185432, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Etheredge, micro-needles are incorporated into a device which drivesby an electrical current the medication through the needles into thetargeted treatment site.

An iontophoresis device has been described by Matsumura et al. and istaught for example in U.S. Pat. No. 7,437,189, the contents of which areincorporated herein by reference in their entirety. According toMatsumura, multiple needles are incorporated into the device which iscapable of delivering ionizable drug into a living body at higher speedor with higher efficiency.

Intradermal delivery of biologically active agents by needle-freeinjection and electroporation has been described by Hoffmann et al andis taught for example in U.S. Pat. No. 7,171,264, the contents of whichare incorporated herein by reference in their entirety. According toHoffmann, one or more needle-free injectors are incorporated into anelectroporation device and the combination of needle-free injection andelectroporation is sufficient to introduce the agent into cells in skin,muscle or mucosa.

A method for electropermeabilization-mediated intracellular delivery hasbeen described by Lundkvist et al. and is taught for example in U.S.Pat. No. 6,625,486, the contents of which are incorporated herein byreference in their entirety. According to Lundkvist, a pair of needleelectrodes is incorporated into a catheter. Said catheter is positionedinto a body lumen followed by extending said needle electrodes topenetrate into the tissue surrounding said lumen. Then the deviceintroduces an agent through at least one of said needle electrodes andapplies electric field by said pair of needle electrodes to allow saidagent pass through the cell membranes into the cells at the treatmentsite.

A delivery system for transdermal immunization has been described byLevin et al. and is taught for example in WO2006003659, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Levin, multiple electrodes are incorporated into the device whichapplies electrical energy between the electrodes to generate microchannels in the skin to facilitate transdermal delivery.

A method for delivering RF energy into skin has been described bySchomacker and is taught for example in WO2011163264, the contents ofwhich are incorporated herein by reference in their entirety. Accordingto Schomacker, multiple needles are incorporated into a device whichapplies vacuum to draw skin into contact with a plate so that needlesinsert into skin through the holes on the plate and deliver RF energy.

VII. Definitions

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C₆ alkyl.

About: As used herein, the term “about” means +/−10% of the recitedvalue.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agentsare administered to a subject at the same time or within an intervalsuch that there may be an overlap of an effect of each agent on thepatient. In some embodiments, they are administered within about 60, 30,15, 10, 5, or 1 minute of one another. In some embodiments, theadministrations of the agents are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Antigens of interest or desired antigens: As used herein, the terms“antigens of interest” or “desired antigens” include those proteins andother biomolecules provided herein that are immunospecifically bound bythe antibodies and fragments, mutants, variants, and alterations thereofdescribed herein. Examples of antigens of interest include, but are notlimited to, insulin, insulin-like growth factor, hGH, tPA, cytokines,such as interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega orIFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF beta,TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety which is capable of or maintains at leasttwo functions. The functions may effect the same outcome or a differentoutcome. The structure that produces the function may be the same ordifferent. For example, bifunctional modified RNAs of the presentinvention may encode a cytotoxic peptide (a first function) while thosenucleosides which comprise the encoding RNA are, in and of themselves,cytotoxic (second function). In this example, delivery of thebifunctional modified RNA to a cancer cell would produce not only apeptide or protein molecule which may ameliorate or treat the cancer butwould also deliver a cytotoxic payload of nucleosides to the cell shoulddegradation, instead of translation of the modified RNA, occur.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments, apolynucleotide, primary construct or mmRNA of the present invention maybe considered biologically active if even a portion of thepolynucleotide, primary construct or mmRNA is biologically active ormimics an activity considered biologically relevant.

Chemical terms: The following provides the definition of variouschemical terms from “acyl” to “thiol.”

The term “acyl,” as used herein, represents a hydrogen or an alkyl group(e.g., a haloalkyl group), as defined herein, that is attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl (i.e., a carboxyaldehyde group), acetyl,propionyl, butanoyl and the like. Exemplary unsubstituted acyl groupsinclude from 1 to 7, from 1 to 11, or from 1 to 21 carbons. In someembodiments, the alkyl group is further substituted with 1, 2, 3, or 4substituents as described herein.

The term “acylamino,” as used herein, represents an acyl group, asdefined herein, attached to the parent molecular group though an aminogroup, as defined herein (i.e., —N(R^(N1))—C(O)—R, where R is H or anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group and R^(N1) isas defined herein). Exemplary unsubstituted acylamino groups includefrom 1 to 41 carbons (e.g., from 1 to 7, from 1 to 13, from 1 to 21,from 2 to 7, from 2 to 13, from 2 to 21, or from 2 to 41 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “acyloxy,” as used herein, represents an acyl group, as definedherein, attached to the parent molecular group though an oxygen atom(i.e., —O—C(O)—R, where R is H or an optionally substituted C₁₋₆, C₁₋₁₀,or C₁₋₂₀ alkyl group). Exemplary unsubstituted acyloxy groups includefrom 1 to 21 carbons (e.g., from 1 to 7 or from 1 to 11 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “alkaryl,” as used herein, represents an aryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkaryl groups arefrom 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, suchas C₁₋₆ alk-C₆₋₁₀ aryl, C₁₋₁₀ alk-C₆₋₁₀ aryl, or C₁₋₂₀ alk-C₆₋₁₀ aryl).In some embodiments, the alkylene and the aryl each can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein forthe respective groups. Other groups preceded by the prefix “alk-” aredefined in the same manner, where “alk” refers to a C₁₋₆ alkylene,unless otherwise noted, and the attached chemical structure is asdefined herein.

The term “alkcycloalkyl” represents a cycloalkyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein (e.g., an alkylene group of from 1 to 4, from 1to 6, from 1 to 10, or form 1 to 20 carbons). In some embodiments, thealkylene and the cycloalkyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 20carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one ormore carbon-carbon double bonds and is exemplified by ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, andthe like. Alkenyls include both cis and trans isomers. Alkenyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from amino, aryl, cycloalkyl, orheterocyclyl (e.g., heteroaryl), as defined herein, or any of theexemplary alkyl substituent groups described herein.

The term “alkenyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkenyl group (e.g., C₂₋₆ or C₂₋₁₀ alkenyl), unlessotherwise specified. Exemplary alkenyloxy groups include ethenyloxy,propenyloxy, and the like. In some embodiments, the alkenyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “alkheteroaryl” refers to a heteroaryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheteroaryl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂heteroaryl, or C₁₋₂₀ alk-C₁₋₁₂ heteroaryl). In some embodiments, thealkylene and the heteroaryl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.Alkheteroaryl groups are a subset of alkheterocyclyl groups.

The term “alkheterocyclyl” represents a heterocyclyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheterocyclyl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heterocyclyl, C₁₋₁₀ alk-C₁₋₁₂heterocyclyl, or C₁₋₂₀ alk-C₁₋₁₂ heterocyclyl). In some embodiments, thealkylene and the heterocyclyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkoxy” represents a chemical substituent of formula —OR,where R is a C₁₋₂₀ alkyl group (e.g., C₁₋₆ or C₁₋₁₀ alkyl), unlessotherwise specified. Exemplary alkoxy groups include methoxy, ethoxy,propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. Insome embodiments, the alkyl group can be further substituted with 1, 2,3, or 4 substituent groups as defined herein (e.g., hydroxy or alkoxy).

The term “alkoxyalkoxy” represents an alkoxy group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkoxy groupsinclude between 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20carbons, such as C₁₋₆ alkoxy-C₁₋₆ alkoxy, C₁₋₁₀ alkoxy-C₁₋₁₀ alkoxy, orC₁₋₂₀ alkoxy-C₁₋₂₀ alkoxy). In some embodiments, the each alkoxy groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkoxyalkyl” represents an alkyl group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups includebetween 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20 carbons,such as C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₁₀ alkoxy-C₁₋₁₀ alkyl, or C₁₋₂₀alkoxy-C₁₋₂₀ alkyl). In some embodiments, the alkyl and the alkoxy eachcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein for the respective group.

The term “alkoxycarbonyl,” as used herein, represents an alkoxy, asdefined herein, attached to the parent molecular group through acarbonyl atom (e.g., —C(O)—OR, where R is H or an optionally substitutedC₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplary unsubstitutedalkoxycarbonyl include from 1 to 21 carbons (e.g., from 1 to 11 or from1 to 7 carbons). In some embodiments, the alkoxy group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein.

The term “alkoxycarbonylalkoxy,” as used herein, represents an alkoxygroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., —O-alkyl-C(O)—OR, where R is anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplaryunsubstituted alkoxycarbonylalkoxy include from 3 to 41 carbons (e.g.,from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31carbons, such as C₁₋₆ alkoxycarbonyl-C₁₋₆ alkoxy, C₁₋₁₀alkoxycarbonyl-C₁₋₁₀ alkoxy, or C₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkoxy). Insome embodiments, each alkoxy group is further independently substitutedwith 1, 2, 3, or 4 substituents, as described herein (e.g., a hydroxygroup).

The term “alkoxycarbonylalkyl,” as used herein, represents an alkylgroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., -alkyl-C(O)—OR, where R is an optionallysubstituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group). Exemplary unsubstitutedalkoxycarbonylalkyl include from 3 to 41 carbons (e.g., from 3 to 10,from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31 carbons, suchas C₁₋₆ alkoxycarbonyl-C₁₋₆ alkyl, C₁₋₁₀ alkoxycarbonyl-C₁₋₁₀ alkyl, orC₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkyl). In some embodiments, each alkyl andalkoxy group is further independently substituted with 1, 2, 3, or 4substituents as described herein (e.g., a hydroxy group).

The term “alkyl,” as used herein, is inclusive of both straight chainand branched chain saturated groups from 1 to 20 carbons (e.g., from 1to 10 or from 1 to 6), unless otherwise specified. Alkyl groups areexemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- andtert-butyl, neopentyl, and the like, and may be optionally substitutedwith one, two, three, or, in the case of alkyl groups of two carbons ormore, four substituents independently selected from the group consistingof: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as definedherein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino(i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉heterocyclyl)oxy; (8)hydroxy; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14)-CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(si)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(si)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein. For example, the alkylene group of aC₁-alkaryl can be further substituted with an oxo group to afford therespective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent asaturated divalent hydrocarbon group derived from a straight or branchedchain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, isopropylene, and the like. The term“C_(x-y) alkylene” and the prefix “C_(x-y) alk-” represent alkylenegroups having between x and y carbons. Exemplary values for x are 1, 2,3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀₅ C₂₋₆₅ C₂₋₁₀₅ orC₂₋₂₀ alkylene). In some embodiments, the alkylene can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein foran alkyl group.

The term “alkylsulfinyl,” as used herein, represents an alkyl groupattached to the parent molecular group through an —S(O)— group.Exemplary unsubstituted alkylsulfinyl groups are from 1 to 6, from 1 to10, or from 1 to 20 carbons. In some embodiments, the alkyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein.

The term “alkylsulfinylalkyl,” as used herein, represents an alkylgroup, as defined herein, substituted by an alkylsulfinyl group.Exemplary unsubstituted alkylsulfinylalkyl groups are from 2 to 12, from2 to 20, or from 2 to 40 carbons. In some embodiments, each alkyl groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bondand is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from aryl, cycloalkyl, or heterocyclyl(e.g., heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

The term “alkynyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkynyl group (e.g., C₂₋₆ or C₂₋₁₀ alkynyl), unlessotherwise specified. Exemplary alkynyloxy groups include ethynyloxy,propynyloxy, and the like. In some embodiments, the alkynyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “amidine,” as used herein, represents a —C(═NH)NH₂ group.

The term “amino,” as used herein, represents —N(R^(N1))₂ wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl, sulfoalkyl,heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g.,alkheteroaryl), wherein each of these recited R^(N1) groups can beoptionally substituted, as defined herein for each group; or two R^(N1)combine to form a heterocyclyl or an N-protecting group, and whereineach R^(N2) is, independently, H, alkyl, or aryl. The amino groups ofthe invention can be an unsubstituted amino (i.e., —NH₂) or asubstituted amino (i.e., —N(R^(N1))₂). In a preferred embodiment, aminois —NH₂ or —NHR^(N1) ₅ wherein R^(N1) is, independently, OH, NO₂, NH₂,NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, carboxyalkyl,sulfoalkyl, or aryl, and each R^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), or C₆₋₁₀ aryl.

The term “amino acid,” as described herein, refers to a molecule havinga side chain, an amino group, and an acid group (e.g., a carboxy groupof —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attachedto the parent molecular group by the side chain, amino group, or acidgroup (e.g., the side chain). In some embodiments, the amino acid isattached to the parent molecular group by a carbonyl group, where theside chain or amino group is attached to the carbonyl group. Exemplaryside chains include an optionally substituted alkyl, aryl, heterocyclyl,alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl.Exemplary amino acids include alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline,ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine,taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groupsmay be optionally substituted with one, two, three, or, in the case ofamino acid groups of two carbons or more, four substituentsindependently selected from the group consisting of: (1) C₁₋₆ alkoxy;(2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxy;(9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(si)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′) R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein.

The term “aminoalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aminoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one or two aromatic rings andis exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl,indanyl, indenyl, and the like, and may be optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from the groupconsisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy;(14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy);(17) —(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, andR^(A′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b)C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18)—(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four andwhere R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (19) —(CH₂)—SO₂R^(D′), where q is an integer from zeroto four and where R^(D′) is selected from the group consisting of (a)alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20)—(CH₂)—SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁-6 alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and(27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can befurther substituted as described herein. For example, the alkylene groupof a C₁-alkaryl or a C₁-alkheterocyclyl can be further substituted withan oxo group to afford the respective aryloyl and (heterocyclyl)oylsubstituent group.

The term “arylalkoxy,” as used herein, represents an alkaryl group, asdefined herein, attached to the parent molecular group through an oxygenatom. Exemplary unsubstituted alkoxyalkyl groups include from 7 to 30carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₆₋₁₀aryl-C₁₋₆ alkoxy, C₆₋₁₀ aryl-C₁₋₁₀ alkoxy, or C₆₋₁₀ aryl-C₁₋₂₀ alkoxy).In some embodiments, the arylalkoxy group can be substituted with 1, 2,3, or 4 substituents as defined herein

The term “aryloxy” represents a chemical substituent of formula —OR′,where R′ is an aryl group of 6 to 18 carbons, unless otherwisespecified. In some embodiments, the aryl group can be substituted with1, 2, 3, or 4 substituents as defined herein.

The term “aryloyl,” as used herein, represents an aryl group, as definedherein, that is attached to the parent molecular group through acarbonyl group. Exemplary unsubstituted aryloyl groups are of 7 to 11carbons. In some embodiments, the aryl group can be substituted with 1,2, 3, or 4 substituents as defined herein.

The term “azido” represents an —N₃ group, which can also be representedas —N═N═N.

The term “bicyclic,” as used herein, refer to a structure having tworings, which may be aromatic or non-aromatic. Bicyclic structuresinclude spirocyclyl groups, as defined herein, and two rings that shareone or more bridges, where such bridges can include one atom or a chainincluding two, three, or more atoms. Exemplary bicyclic groups include abicyclic carbocyclyl group, where the first and second rings arecarbocyclyl groups, as defined herein; a bicyclic aryl groups, where thefirst and second rings are aryl groups, as defined herein; bicyclicheterocyclyl groups, where the first ring is a heterocyclyl group andthe second ring is a carbocyclyl (e.g., aryl) or heterocyclyl (e.g.,heteroaryl) group; and bicyclic heteroaryl groups, where the first ringis a heteroaryl group and the second ring is a carbocyclyl (e.g., aryl)or heterocyclyl (e.g., heteroaryl) group. In some embodiments, thebicyclic group can be substituted with 1, 2, 3, or 4 substituents asdefined herein for cycloalkyl, heterocyclyl, and aryl groups.

The terms “carbocyclic” and “carbocyclyl,” as used herein, refer to anoptionally substituted C₃₁₂ monocyclic, bicyclic, or tricyclic structurein which the rings, which may be aromatic or non-aromatic, are formed bycarbon atoms. Carbocyclic structures include cycloalkyl, cycloalkenyl,and aryl groups.

The term “carbamoyl,” as used herein, represents —C(O)—N(R^(N1))₂, wherethe meaning of each R^(N1) is found in the definition of “amino”provided herein.

The term “carbamoylalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carbamoyl group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “carbamyl,” as used herein, refers to a carbamate group havingthe structure —NR^(N1)C(═O)OR or —OC(═O)N(R^(N1))₂, where the meaning ofeach R^(N1) is found in the definition of “amino” provided herein, and Ris alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl (e.g.,heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as definedherein.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxyaldehyde” represents an acyl group having the structure—CHO.

The term “carboxy,” as used herein, means —CO₂H.

The term “carboxyalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkoxy group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein for the alkyl group.

The term “carboxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkoxy” represents a chemical substituent of formula —OR,where R is a C₃₋₈ cycloalkyl group, as defined herein, unless otherwisespecified. The cycloalkyl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein. Exemplary unsubstitutedcycloalkoxy groups are from 3 to 8 carbons. In some embodiment, thecycloalkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to eightcarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl,and the like. When the cycloalkyl group includes one carbon-carbondouble bond, the cycloalkyl group can be referred to as a “cycloalkenyl”group. Exemplary cycloalkenyl groups include cyclopentenyl,cyclohexenyl, and the like. The cycloalkyl groups of this invention canbe optionally substituted with: (1) C₁₋₇ acyl (e.g., carboxyaldehyde);(2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl,(carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g., perfluoroalkyl),hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3)C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such as perfluoroalkoxy); (4) C₁₋₆alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8)azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo;(12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g.,C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q is an integer fromzero to four, and R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl;(18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to fourand where R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and (d)C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integerfrom zero to four and where R^(D′) is selected from the group consistingof (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) C₂₋₂₀alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of thesegroups can be further substituted as described herein. For example, thealkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The term “diastereomer,” as used herein means stereoisomers that are notmirror images of one another and are non-superimposable on one another.

The term “effective amount” of an agent, as used herein, is that amountsufficient to effect beneficial or desired results, for example,clinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. For example, in the context ofadministering an agent that treats cancer, an effective amount of anagent is, for example, an amount sufficient to achieve treatment, asdefined herein, of cancer, as compared to the response obtained withoutadministration of the agent.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound of the invention, having an optical purity orenantiomeric excess (as determined by methods standard in the art) of atleast 80% (i.e., at least 90% of one enantiomer and at most 10% of theother enantiomer), preferably at least 90% and more preferably at least98%.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

The term “haloalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkoxy may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkoxy groupsinclude perfluoroalkoxys (e.g., —OCF₃), —OCHF₂, —OCH₂F, —OCCl₃,—OCH₂CH₂Br, —OCH₂CH(CH₂CH₂Br)CH₃, and —OCHICH₃. In some embodiments, thehaloalkoxy group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “haloalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkyl may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkyl groupsinclude perfluoroalkyls (e.g., —CF₃), —CHF₂, —CH₂F, —CCl₃, —CH₂CH₂Br,—CH₂CH(CH₂CH₂Br)CH₃, and —CHICH₃. In some embodiments, the haloalkylgroup can be further substituted with 1, 2, 3, or 4 substituent groupsas described herein for alkyl groups.

The term “heteroalkylene,” as used herein, refers to an alkylene group,as defined herein, in which one or two of the constituent carbon atomshave each been replaced by nitrogen, oxygen, or sulfur. In someembodiments, the heteroalkylene group can be further substituted with 1,2, 3, or 4 substituent groups as described herein for alkylene groups.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. Exemplaryunsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10,1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heterocyclyl,” as used herein represents a 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl and the like. Examples of fused heterocyclyls includetropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics includepyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl,morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, benzothienyl, and the like, including dihydro andtetrahydro forms thereof, where one or more double bonds are reduced andreplaced with hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl; 2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl; 3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7 H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1 H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups alsoinclude groups of the formula

where

E′ is selected from the group consisting of —N— and —CH—; F′ is selectedfrom the group consisting of —N═CH—, —NH—CH₂—, —NH—C(O)—, —NH—, —CH═N—,—CH₂—NH—, —C(O)—NH—, —CH═CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—, —O—, and—S—; and G′ is selected from the group consisting of —CH— and —N—. Anyof the heterocyclyl groups mentioned herein may be optionallysubstituted with one, two, three, four or five substituentsindependently selected from the group consisting of: (1) C₁₋₇ acyl(e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl,azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zeroto four and where each of R^(E′) and R^(F′) is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈ cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl(e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In someembodiments, each of these groups can be further substituted asdescribed herein. For example, the alkylene group of a C₁-alkaryl or aC₁-alkheterocyclyl can be further substituted with an oxo group toafford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “(heterocyclyl)imino,” as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through an imino group. In some embodiments, the heterocyclylgroup can be substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “(heterocyclyl)oxy,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group throughan oxygen atom. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “(heterocyclyl)oyl,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group througha carbonyl group. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “hydrocarbon,” as used herein, represents a group consistingonly of carbon and hydrogen atoms.

The term “hydroxy,” as used herein, represents an —OH group.

The term “hydroxyalkenyl,” as used herein, represents an alkenyl group,as defined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by dihydroxypropenyl,hydroxyisopentenyl, and the like.

The term “hydroxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by hydroxymethyl,dihydroxypropyl, and the like.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound of the invention. It isrecognized that the compounds of the invention can have one or morechiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached one or two N-protecting groups,as defined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups, such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

The term “nitro,” as used herein, represents an —NO₂ group.

The term “oxo” as used herein, represents ═O.

The term “perfluoroalkyl,” as used herein, represents an alkyl group, asdefined herein, where each hydrogen radical bound to the alkyl group hasbeen replaced by a fluoride radical. Perfluoroalkyl groups areexemplified by trifluoromethyl, pentafluoroethyl, and the like.

The term “perfluoroalkoxy,” as used herein, represents an alkoxy group,as defined herein, where each hydrogen radical bound to the alkoxy grouphas been replaced by a fluoride radical. Perfluoroalkoxy groups areexemplified by trifluoromethoxy, pentafluoroethoxy, and the like.

The term “spirocyclyl,” as used herein, represents a C₂₋₇ alkylenediradical, both ends of which are bonded to the same carbon atom of theparent group to form a spirocyclic group, and also a C₁₋₆ heteroalkylenediradical, both ends of which are bonded to the same atom. Theheteroalkylene radical forming the spirocyclyl group can containing one,two, three, or four heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. In some embodiments, thespirocyclyl group includes one to seven carbons, excluding the carbonatom to which the diradical is attached. The spirocyclyl groups of theinvention may be optionally substituted with 1, 2, 3, or 4 substituentsprovided herein as optional substituents for cycloalkyl and/orheterocyclyl groups.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

The term “sulfoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a sulfo group of —SO₃H. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thioalkaryl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkaryl group. In someembodiments, the alkaryl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein.

The term “thioalkheterocyclyl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkheterocyclyl group. In someembodiments, the alkheterocyclyl group can be further substituted with1, 2, 3, or 4 substituent groups as described herein.

The term “thioalkoxy,” as used herein, represents a chemical substituentof formula —SR, where R is an alkyl group, as defined herein. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “thiol” represents an —SH group.

Compound: As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone—enol pairs, amide—imidicacid pairs, lactam—lactim pairs, amide—imidic acid pairs, enamine—iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an oligonucleotide orpolypeptide or may apply to a portion, region or feature thereof.

Controlled Release: As used herein, the term “controlled release” refersto a pharmaceutical composition or compound release profile thatconforms to a particular pattern of release to effect a therapeuticoutcome.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits. Cyclic molecules suchas the engineered RNA or mRNA of the present invention may be singleunits or multimers or comprise one or more components of a complex orhigher order structure.

Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing,suppressing the growth, division, or multiplication of a cell (e.g., amammalian cell (e.g., a human cell)), bacterium, virus, fungus,protozoan, parasite, prion, or a combination thereof.

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery of apolynucleotide, primary construct or mmRNA to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels may be located at any position in thepeptides or proteins disclosed herein. They may be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Dosing regimen: As used herein, a “dosing regimen” is a schedule ofadministration or physician determined regimen of treatment,prophylaxis, or palliative care.

Dose splitting factor (DSF)-ratio of PUD of dose split treatment dividedby PUD of total daily dose or single unit dose. The value is derivedfrom comparison of dosing regimens groups.

Encapsulate: As used herein, the term “encapsulate” means to enclose,surround or encase.

Encoded protein cleavage signal: As used herein, “encoded proteincleavage signal” refers to the nucleotide sequence which encodes aprotein cleavage signal.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Exosome: As used herein, “exosome” is a vesicle secreted by mammaliancells or a complex involved in RNA degradation.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least apolynucleotide, primary construct or mmRNA and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the invention, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the invention, two protein sequences are considered tobe homologous if the proteins are at least about 50%, 60%, 70%, 80%, or90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between oligonucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% by weight of thecompound of the present disclosure, or salt thereof. Methods forisolating compounds and their salts are routine in the art.

Linker: As used herein, a linker refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., a detectable or therapeutic agent, at asecond end. The linker may be of sufficient length as to not interferewith incorporation into a nucleic acid sequence. The linker can be usedfor any useful purpose, such as to form mmRNA multimers (e.g., throughlinkage of two or more polynucleotides, primary constructs, or mmRNAmolecules) or mmRNA conjugates, as well as to administer a payload, asdescribed herein. Examples of chemical groups that can be incorporatedinto the linker include, but are not limited to, alkyl, alkenyl,alkynyl, amido, amino, ether, thioether, ester, alkylene,heteroalkylene, aryl, or heterocyclyl, each of which can be optionallysubstituted, as described herein. Examples of linkers include, but arenot limited to, unsaturated alkanes, polyethylene glycols (e.g.,ethylene or propylene glycol monomeric units, e.g., diethylene glycol,dipropylene glycol, triethylene glycol, tripropylene glycol,tetraethylene glycol, or tetraethylene glycol), and dextran polymers andderivatives thereof. Other examples include, but are not limited to,cleavable moieties within the linker, such as, for example, a disulfidebond (—S—S—) or an azo bond (—N═N—), which can be cleaved using areducing agent or photolysis. Non-limiting examples of a selectivelycleavable bond include an amido bond can be cleaved for example by theuse of tris(2-carboxyethyl)phosphine (TCEP), or other reducing agents,and/or photolysis, as well as an ester bond can be cleaved for exampleby acidic or basic hydrolysis.

MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA)binding site represents a nucleotide location or region of a nucleicacid transcript to which at least the “seed” region of a miRNA binds.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the invention. Molecules may be modified inmany ways including chemically, structurally, and functionally. In oneembodiment, the mRNA molecules of the present invention are modified bythe introduction of non-natural nucleosides and/or nucleotides, e.g., asit relates to the natural ribonucleotides A, U, G, and C. Noncanonicalnucleotides such as the cap structures are not considered “modified”although they differ from the chemical structure of the A, C, G, Uribonucleotides.

Mucus: As used herein, “mucus” refers to the natural substance that isviscous and comprises mucin glycoproteins.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Non-human vertebrate: As used herein, a “non human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Optionally substituted: Herein a phrase of the form “optionallysubstituted X” (e.g., optionally substituted alkyl) is intended to beequivalent to “X, wherein X is optionally substituted” (e.g., “alkyl,wherein said alkyl is optionally substituted”). It is not intended tomean that the feature “X” (e.g. alkyl) per se is optional.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Paratope: As used herein, a “paratope” refers to the antigen-bindingsite of an antibody.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. The pharmaceutically acceptable salts of the presentdisclosure include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, andUse, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge etal., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of whichis incorporated herein by reference in its entirety.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the inventionwherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Polypeptide per unit drug (PUD): As used herein, a PUD or product perunit drug, is defined as a subdivided portion of total daily dose,usually 1 mg, pg, kg, etc., of a product (such as a polypeptide) asmeasured in body fluid or tissue, usually defined in concentration suchas pmol/mL, mmol/mL, etc divided by the measure in the body fluid.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may by covalently bonded or sequestered in some wayand which release or are converted into the active drug moiety prior to,upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Protein cleavage site: As used herein, “protein cleavage site” refers toa site where controlled cleavage of the amino acid chain can beaccomplished by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein “protein cleavage signal” refersto at least one amino acid that flags or marks a polypeptide forcleavage.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Pseudouridine: As used herein, pseudouridine refers to the C-glycosideisomer of the nucleoside uridine. A “pseudouridine analog” is anymodification, variant, isoform or derivative of pseudouridine. Forexample, pseudouridine analogs include but are not limited to1-carboxymethyl-pseudouridine, 1-propynyl-pseudouridine,1-taurinomethyl-pseudouridine, 1-taurinomethyl-4-thio-pseudouridine,1-methylpseudouridine (m¹ψ), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,N1-methyl-pseudouridine,1-methyl-3-β-amino-3-carboxypropyl)pseudouridine (acp³ψ), and2′-O-methyl-pseudouridine (ψm).

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Signal Sequences: As used herein, the phrase “signal sequences” refersto a sequence which can direct the transport or localization of aprotein.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administed in one dose/at one time/single route/single pointof contact, i.e., single administration event.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Sustained release: As used herein, the term “sustained release” refersto a pharmaceutical composition or compound release profile thatconforms to a release rate over a specific period of time.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES Example 1 Modified mRNA Production

Modified mRNAs (mmRNA) according to the invention may be made usingstandard laboratory methods and materials. The open reading frame (ORF)of the gene of interest may be flanked by a 5′ untranslated region (UTR)which may contain a strong Kozak translational initiation signal and/oran alpha-globin 3′ UTR which may include an oligo(dT) sequence fortemplated addition of a poly-A tail. The modified mRNAs may be modifiedto reduce the cellular innate immune response. The modifications toreduce the cellular response may include pseudouridine (ψ) and5-methyl-cytidine (5meC, 5mc or m⁵C). (See, Kariko K et al. Immunity23:165-75 (2005), Kariko K et al. Mol Ther 16:1833-40 (2008), Anderson BR et al. NAR (2010); each of which are herein incorporated by referencein their entireties).

The ORF may also include various upstream or downstream additions (suchas, but not limited to, β-globin, tags, etc.) may be ordered from anoptimization service such as, but limited to, DNA2.0 (Menlo Park,Calif.) and may contain multiple cloning sites which may have XbaIrecognition. Upon receipt of the construct, it may be reconstituted andtransformed into chemically competent E. coli.

For the present invention, NEB DH5-alpha Competent E. coli are used.Transformations are performed according to NEB instructions using 100 ngof plasmid.

The protocol is as follows:

-   -   1 Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for        10 minutes.    -   2 Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell        mixture. Carefully flick the tube 4-5 times to mix cells and        DNA. Do not vortex.    -   3 Place the mixture on ice for 30 minutes. Do not mix.    -   4 Heat shock at 42° C. for exactly 30 seconds. Do not mix.    -   5 Place on ice for 5 minutes. Do not mix.    -   6 Pipette 950 μl of room temperature SOC into the mixture.    -   7 Place at 37° C. for 60 minutes. Shake vigorously (250 rpm) or        rotate.    -   8 Warm selection plates to 37° C.    -   9 Mix the cells thoroughly by flicking the tube and inverting.

Spread 50-100 μl of each dilution onto a selection plate and incubateovernight at 37° C. Alternatively, incubate at 30° C. for 24-36 hours or25° C. for 48 hours.

A single colony is then used to inoculate 5 ml of LB growth media usingthe appropriate antibiotic and then allowed to grow (250 RPM, 37° C.)for 5 hours. This is then used to inoculate a 200 ml culture medium andallowed to grow overnight under the same conditions.

To isolate the plasmid (up to 850 μg), a maxi prep is performed usingthe Invitrogen PURELINK™ HiPure Maxiprep Kit (Carlsbad, Calif.),following the manufacturer's instructions.

In order to generate cDNA for In Vitro Transcription (IVT), the plasmid(an Example of which is shown in FIG. 3) is first linearized using arestriction enzyme such as XbaI. A typical restriction digest with XbaIwill comprise the following: Plasmid 1.0 μg; 10× Buffer 1.0 μl; XbaI1.5μl; dH₂0 up to 10 μl; incubated at 37° C. for 1 hr. If performing atlab scale (<5 μg), the reaction is cleaned up using Invitrogen'sPURELINK™ PCR Micro Kit (Carlsbad, Calif.) per manufacturer'sinstructions. Larger scale purifications may need to be done with aproduct that has a larger load capacity such as Invitrogen's standardPURELINK™ PCR Kit (Carlsbad, Calif.). Following the cleanup, thelinearized vector is quantified using the NanoDrop and analyzed toconfirm linearization using agarose gel electrophoresis.

The methods described herein to make modified mRNA may be used toproduce molecules of all sizes including long molecules. Modified mRNAusing the described methods has been made for different sized moleculesincluding glucosidase, alpha; acid (GAA) (3.2 kb), cystic fibrosistransmembrane conductance regulator (CFTR) (4.7 kb), Factor VII (7.3kb), lysosomal acid lipase (45.4 kDa), glucocerebrosidase (59.7 kDa) andiduronate 2-sulfatase (76 kDa).

As a non-limiting example, G-CSF may represent the polypeptide ofinterest. Sequences used in the steps outlined in Examples 1-5 are shownin Table 11. It should be noted that the start codon (ATG) has beenunderlined in each sequence of Table 11.

TABLE 11 G-CSF Sequences SEQ ID NO Description 21435 cDNA sequence:ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGA 21436 cDNA having T7polymerase site, AfeI and Xba restriction site: TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA 21437 Optimized sequence; containing T7polymerase site, AfeI and Xba restriction site TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA 21438 mRNA sequence (transcribed)GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCA CCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACA UCUUGCGCAGCCGUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGG AAG

Example 2 PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2× KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2× KAPA ReadyMix 12.5 μl; Forward Primer (10 uM) 0.75 μl;Reverse Primer (10 uM) 0.75 μl; Template cDNA 100 ng; and dH₂0 dilutedto 25.0 μl. The reaction conditions are at 95° C. for 5 min. and 25cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45sec, then 72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant invention incorporates a poly-T₁₂₀ fora poly-A₁₂₀ in the mRNA. Other reverse primers with longer or shorterpoly(T) tracts can be used to adjust the length of the poly(A) tail inthe mRNA.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NANODROP™ andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 3 In vitro Transcription (IVT)

The in vitro transcription reaction generates mRNA containing modifiednucleotides or modified RNA. The input nucleotide triphosphate (NTP) mixis made in-house using natural and un-natural NTPs.

A typical in vitro transcription reaction includes the following:

1 Template cDNA 1.0 μg 2 10x transcription buffer (400 mM Tris- 2.0 μlHCl pH 8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 3 Custom NTPs (25mM each) 7.2 μl 4 RNase Inhibitor 20 U 5 T7 RNA polymerase 3000 U 6 dH₂0Up to 20.0 μl. and 7 Incubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 4 Enzymatic Capping of mRNA

Capping of the mRNA is performed as follows where the mixture includes:IVT RNA 60 μg-180 μg and dH₂0 up to 72 μl. The mixture is incubated at65° C. for 5 minutes to denature RNA, and then is transferredimmediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂0 (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The mRNA is then purified using Ambion's MEGACLEAR™ Kit (Austin, Tex.)following the manufacturer's instructions. Following the cleanup, theRNA is quantified using the NANODROP™ (ThermoFisher, Waltham, Mass.) andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred. The RNA productmay also be sequenced by running a reverse-transcription-PCR to generatethe cDNA for sequencing.

Example 5 PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymeraseis preferably a recombinant enzyme expressed in yeast.

For studies performed and described herein, the poly-A tail is encodedin the IVT template to comprise 160 nucleotides in length. However, itshould be understood that the processivity or integrity of the polyAtailing reaction may not always result in exactly 160 nucleotides. HencepolyA tails of approximately 160 nucleotides, e.g, about 150-165, 155,156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scopeof the invention.

Example 6 Natural 5′ Caps and 5′ Cap Analogues

-capping of modified RNA may be completed concomitantly during the invitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3″-O-Me-m7G(5′)ppp(5′) G [the ARCAcap];G(5)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 7 Capping

A. Protein Expression Assay

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 21435;mRNA sequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 21438with a polyA tail approximately 160 nucletodies in length not shown insequence) containing the ARCA (3′ O-Me-m7G(5′)ppp(5′)G) cap analog orthe Cap1 structure can be transfected into human primary keratinocytesat equal concentrations. 6, 12, 24 and 36 hours post-transfection theamount of G-CSF secreted into the culture medium can be assayed byELISA. Synthetic mRNAs that secrete higher levels of G-CSF into themedium would correspond to a synthetic mRNA with a highertranslationally-competent Cap structure.

B. Purity Analysis Synthesis

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 21435;mRNA sequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 21438with a polyA tail approximately 160 nucletodies in length not shown insequence) containing the ARCA cap analog or the Cap1 structure crudesynthesis products can be compared for purity using denaturingAgarose-Urea gel electrophoresis or HPLC analysis. Synthetic mRNAs witha single, consolidated band by electrophoresis correspond to the higherpurity product compared to a synthetic mRNA with multiple bands orstreaking bands. Synthetic mRNAs with a single HPLC peak would alsocorrespond to a higher purity product. The capping reaction with ahigher efficiency would provide a more pure mRNA population.

C. Cytokine Analysis

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 21435;mRNA sequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 21438with a polyA tail approximately 160 nucletodies in length not shown insequence) containing the ARCA cap analog or the Cap1 structure can betransfected into human primary keratinocytes at multiple concentrations.6, 12, 24 and 36 hours post-transfection the amount of pro-inflammatorycytokines such as TNF-alpha and IFN-beta secreted into the culturemedium can be assayed by ELISA. Synthetic mRNAs that secrete higherlevels of pro-inflammatory cytokines into the medium would correspond toa synthetic mRNA containing an immune-activating cap structure.

D. Capping Reaction Efficiency

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 21435;mRNA sequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 21438with a polyA tail approximately 160 nucletodies in length not shown insequence) containing the ARCA cap analog or the Cap1 structure can beanalyzed for capping reaction efficiency by LC-MS after capped mRNAnuclease treatment. Nuclease treatment of capped mRNAs would yield amixture of free nucleotides and the capped 5′-5-triphosphate capstructure detectable by LC-MS. The amount of capped product on the LC-MSspectra can be expressed as a percent of total mRNA from the reactionand would correspond to capping reaction efficiency. The cap structurewith higher capping reaction efficiency would have a higher amount ofcapped product by LC-MS.

Example 8 Agarose Gel Electrophoresis of Modified RNA or RT PCR Products

Individual modified RNAs (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) are loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes according to the manufacturer protocol.

Example 9 Nanodrop Modified RNA Quantification and UV Spectral Data

Modified RNAs in TE buffer (1 μl) are used for Nanodrop UV absorbancereadings to quantitate the yield of each modified RNA from an in vitrotranscription reaction.

Example 10 Formulation of Modified mRNA Using Lipidoids

Modified mRNAs (mmRNA) are formulated for in vitro experiments by mixingthe mmRNA with the lipidoid at a set ratio prior to addition to cells.In vivo formulation may require the addition of extra ingredients tofacilitate circulation throughout the body. To test the ability of theselipidoids to form particles suitable for in vivo work, a standardformulation process used for siRNA-lipidoid formulations was used as astarting point. Initial mmRNA-lipidoid formulations may consist ofparticles composed of 42% lipidoid, 48% cholesterol and 10% PEG, withfurther optimization of ratios possible. After formation of theparticle, mmRNA is added and allowed to integrate with the complex. Theencapsulation efficiency is determined using a standard dye exclusionassays.

Materials and Methods for Examples 11-15 A. Lipid Synthesis

Six lipids, DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 andDLin-MC3-DMA, were synthesized by methods outlined in the art in orderto be formulated with modified RNA. DLin-DMA and precursorsweresynthesized as described in Heyes et. al, J. Control Release, 2005, 107,276-287. DLin-K-DMA and DLin-KC2-DMA and precursors were synthesized asdescribed in Semple et. al, Nature Biotechnology, 2010, 28, 172-176.98N12-5 and precursor were synthesized as described in Akinc et. al,Nature Biotechnology, 2008, 26, 561-569.

C12-200 and precursors were synthesized according to the method outlinedin Love et. al, PNAS, 2010, 107, 1864-1869. 2-epoxydodecane (5.10 g,27.7 mmol, 8.2 eq) was added to a vial containing Amine 200 (0.723 g,3.36 mmol, 1 eq) and a stirring bar. The vial was sealed and warmed to80° C. The reaction was stirred for 4 days at 80° C. Then the mixturewas purified by silica gel chromatography using a gradient from puredichloromethane (DCM) to DCM:MeOH 98:2. The target compound was furtherpurified by RP-HPLC to afford the desired compound.

DLin-MC3-DMA and precursors were synthesized according to proceduresdescribed in WO 2010054401 herein incorporated by reference in itsentirety. A mixture of dilinoleyl methanol (1.5 g, 2.8 mmol, 1 eq),N,N-dimethylaminobutyric acid (1.5 g, 2.8 mmol, 1 eq), DIPEA (0.73 mL,4.2 mmol, 1.5 eq) and TBTU(1.35 g, 4.2 mmol, 1.5 eq) in 10 mL of DMF wasstirred for 10 h at room temperature. Then the reaction mixture wasdiluted in ether and washed with water. The organic layer was dried overanhydrous sodium sulfate, filtrated and concentrated under reducedpressure. The crude product was purified by silica gel chromatographyusing a gradient DCM to DCM:MeOH 98:2. Subsequently the target compoundwas subjected to an additional RP-HPLC purification which was done usinga YMC—Pack C4 column to afford the target compound.

B. Formulation of Modified RNA Nanoparticles

Solutions of synthesized lipid, 1,2-distearoyl-3-phosphatidylcholine(DSPC) (Avanti Polar Lipids, Alabaster, Ala.), cholesterol(Sigma-Aldrich, Taufkirchen, Germany), andα-[3′-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-w-methoxy-polyoxyethylene(PEG-c-DOMG) (NOF, Bouwelven, Belgium) were prepared at concentrationsof 50 mM in ethanol and stored at −20° C. The lipids were combined toyield molar ratio of 50:10:38.5:1.5 (Lipid: DSPC: Cholesterol:PEG-c-DOMG) and diluted with ethanol to a final lipid concentration of25 mM. Solutions of modified mRNA at a concentration of 1-2 mg/mL inwater were diluted in 50 mM sodium citrate buffer at a pH of 3 to form astock modified mRNA solution. Formulations of the lipid and modifiedmRNA were prepared by combining the synthesized lipid solution with themodified mRNA solution at total lipid to modified mRNA weight ratio of10:1, 15:1, 20:1 and 30:1. The lipid ethanolic solution was rapidlyinjected into aqueous modified mRNA solution to afford a suspensioncontaining 33% ethanol. The solutions were injected either manually (MI)or by the aid of a syringe pump (SP) (Harvard Pump 33 Dual Syringe PumpHarvard Apparatus Holliston, Mass.).

To remove the ethanol and to achieve the buffer exhange, theformulations were dialyzed twice against phosphate buffered saline(PBS), pH 7.4 at volumes 200-times of the primary product using aSlide-A-Lyzer cassettes (Thermo Fisher Scientific Inc. Rockford, Ill.)with a molecular weight cutoff (MWCO) of 10 kD. The first dialysis wascarried at room temperature for 3 hours and then the formulations weredialyzed overnight at 4° C. The resulting nanoparticle suspension wasfiltered through 0.2 μm sterile filter (Sarstedt, Nümbrecht, Germany)into glass vials and sealed with a crimp closure.

C. Characterization of Formulations

A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) was used to determine the particle size, the polydispersity index(PDI) and the zeta potential of the modified mRNA nanoparticles in 1×PBS in determining particle size and 15 mM PBS in determining zetapotential.

Ultraviolet-visible spectroscopy was used to determine the concentrationof modified mRNA nanoparticle formulation. 100 μL of the dilutedformulation in 1× PBS was added to 900 μL of a 4:1 (v/v) mixture ofmethanol and chloroform. After mixing, the absorbance spectrum of thesolution was recorded between 230 nm and 330 nm on a DU 800spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea,Calif.). The modified RNA concentration in the nanoparicle formulationwas calculated based on the extinction coefficient of the modified RNAused in the formulation and on the difference between the absorbance ata wavelength of 260 nm and the baseline value at a wavelength of 330 nm.

QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.)was used to evaluate the encapsulation of modified RNA by thenanoparticle. The samples were diluted to a concentration ofapproximately 5 μg/mL in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5).50 μL of the diluted samples were transferred to a polystyrene 96 wellplate, then either 50 μL of TE buffer or 50 μL of a 2% Triton X-100solution was added. The plate was incubated at a temperature of 37° C.for 15 minutes. The RIBOGREEN® reagent was diluted 1:100 in TE buffer,100 μL of this solution was added to each well. The fluorescenceintensity was measured using a fluorescence plate reader (Wallac Victor1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.) at an excitationwavelength of ˜480 nm and an emission wavelength of ˜520 nm. Thefluorescence values of the reagent blank were subtracted from that ofeach of the samples and the percentage of free modified RNA wasdetermined by dividing the fluorescence intensity of the intact sample(without addition of Triton X-100) by the fluorescence value of thedisrupted sample (caused by the addition of Triton X-100).

D. In Vitro Incubation

Human embryonic kidney epithelial (HEK293) and hepatocellular carcinomaepithelial (HepG2) cells (LGC standards GmbH, Wesel, Germany) wereseeded on 96-well plates (Greiner Bio-one GmbH, Frickenhausen, Germany)and plates for HEK293 cells were precoated with collagen type1. HEK293were seeded at a density of 30,000 and HepG2 were seeded at a density of35,000 cells per well in 100 μl cell culture medium. For HEK293 the cellculture medium was DMEM, 10% FCS, adding 2 mM L-Glutamine, 1 mMSodiumpyruvate and 1× non-essential amino acids (Biochrom AG, Berlin,Germany) and 1.2 mg/ml Sodiumbicarbonate (Sigma-Aldrich, Munich,Germany) and for HepG2 the culture medium was MEM (Gibco LifeTechnologies, Darmstadt, Germany), 10% FCS adding 2 mM L-Glutamine, 1 mMSodiumpyruvate and 1× non-essential amino acids (Biochrom AG, Berlin,Germany. Formulations containing mCherry mRNA (mRNA sequence shown inSEQ ID NO: 21439; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1) were added in quadruplicates directly afterseeding the cells and incubated. The mCherry cDNA with the T7 promoter,5′ untranslated region (UTR) and 3′ UTR used in in vitro transcription(IVT) is given in SEQ ID NO: 21440. The mCherry mRNA was modified with a5meC at each cytosine and pseudouridine replacement at each uridinesite.

Cells were harvested by transferring the culture media supernatants to a96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg,Germany). Cells were trypsinized with ½ volume Trypsin/EDTA (BiochromAG, Berlin, Germany), pooled with respective supernatants and fixed byadding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5%formaldehyde (Merck, Darmstadt, Germany). Samples then were submitted toa flow cytometer measurement with a 532 nm excitation laser and the610/20 filter for PE-Texas Red in a LSRII cytometer (Beckton DickinsonGmbH, Heidelberg, Germany). The mean fluorescence intensity (MFI) of allevents and the standard deviation of four independent wells arepresented in for samples analyzed.

Example 11 Purification of Nanoparticle Formulations

Nanoparticle formulations of DLin-KC2-DMA and 98N12-5 in HEK293 andHepG2 were tested to determine if the mean fluorescent intensity (MFI)was dependent on the lipid to modified RNA ratio and/or purification.Three formulations of DLin-KC2-DMA and two formulations of 98N12-5 wereproduced using a syringe pump to the specifications described in Table12. Purified samples were purified by SEPHADEX™ G-25 DNA grade (GEHealthcare, Sweden). Each formulation before and after purification (aP)was tested at concentration of 250 ng modified RNA per well in a 24 wellplate. The percentage of cells that are positive for the marker for FL4channel (% FL4-positive) when analyzed by the flow cytometer for eachformulation and the background sample, and the MFI of the marker for theFL4 channel for each formulation and the background sample are shown inTable 13. The formulations which had been purified had a slightly higherMFI than those formulations tested before purification.

TABLE 12 Formulations Formulation # Lipid Lipid/RNA wt/wt Mean size (nm)NPA-001-1 DLin-KC2-DMA 10 155 nm PDI: 0.08 NPA-001-1 aP DLin-KC2-DMA 10141 nm PDI: 0.14 NPA-002-1 DLin-KC2-DMA 15 140 nm PDI: 0.11 NPA-002-1 aPDLin-KC2-DMA 15 125 nm PDI: 0.12 NPA-003-1 DLin-KC2-DMA 20 114 nm PDI:0.08 NPA-003-1 aP DLin-KC2-DMA 20 104 nm PDI: 0.06 NPA-005-1 98N12-5 15127 nm PDI: 0.12 NPA-005-1 aP 98N12-5 15 134 nm PDI: 0.17 NPA-006-198N12 20 126 nm PDI: 0.08 NPA-006-1 aP 98N12 20 118 nm PDI: 0.13

TABLE 13 HEK293 and HepG2, 24-well, 250 ng Modified RNA/well %FL4-positive FL4 MFI Formulation HEK293 HepG2 HEK293 HepG2 Untreated0.33 0.40 0.25 0.30 NPA-001-1 62.42 5.68 1.49 0.41 NPA-001-ap 87.32 9.023.23 0.53 NPA-002-1 91.28 9.90 4.43 0.59 NPA-002-ap 92.68 14.02 5.070.90 NPA-003-1 87.70 11.76 6.83 0.88 NPA-003-ap 88.88 15.46 8.73 1.06NPA-005-1 50.60 4.75 1.83 0.46 NPA-005-ap 38.64 5.16 1.32 0.46 NPA-006-154.19 13.16 1.30 0.60 NPA-006-ap 49.97 13.74 1.27 0.61

Example 12 Concentration Response Curve

Nanoparticle formulations of 98N12-5 (NPA-005) and DLin-KC2-DMA(NPA-003) were tested at varying concentrations to determine the MFI ofFL4 or mCherry (mRNA sequence shown in SEQ ID NO: 21439; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) over a range of doses.The formulations tested are outlined in Table 14. To determine theoptimal concentration of nanoparticle formulations of 98N12-5, varyingconcentrations of formulated modified RNA (100 ng, 10 ng, 1.0 ng, 0.1 ngand 0.01 ng per well) were tested in a 24-well plate of HEK293, and theresults of the FL4 MFI of each dose are shown in Table 15. Likewise, todetermine the optimal concentration of nanoparticle formulations ofDLin-KC2-DMA, varying concentrations of formulated modified RNA (250 ng100 ng, 10 ng, 1.0 ng, 0.1 ng and 0.01 ng per well) were tested in a24-well plate of HEK293, and the results of the FL4 MFI of each dose areshown in Table 16. Nanoparticle formulations of DLin-KC2-DMA were alsotested at varying concentrations of formulated modified RNA (250 ng, 100ng and 30 ng per well) in a 24 well plate of HEK293, and the results ofthe FL4 MFI of each dose are shown in Table 17. A dose of 1 ng/well for98N12-5 and a dose of 10 ng/well for DLin-KC2-DMA were found to resemblethe FL4 MFI of the background.

To determine how close the concentrations resembled the background, weutilized a flow cytometer with optimized filter sets for detection ofmCherry expression, and were able to obtain results with increasedsensitivity relative to background levels. Doses of 25 ng/well, 0.25ng/well, 0.025 ng/well and 0.0025 ng/well were analyzed for 98N12-5(NPA-005) and DLin-KC2-DMA (NPA-003) to determine the MFI of mCherry. Asshown in Table 18, the concentration of 0.025 ng/well and lesserconcentrations are similar to the background MFI level of mCherry whichis about 386.125.

TABLE 14 Formulations Formulation # NPA-003 NPA-005 Lipid DLin-KC2-DMA98N12-5 Lipid/RNA 20 15 wt/wt Mean size 114 nm 106 nm PDI: 0.08 PDI:0.12

TABLE 15 HEK293, NPA-005, 24-well, n = 4 Formulation FL4 MFI Untreatedcontrol 0.246 NPA-005 100 ng 2.2175 NPA-005 10 ng 0.651 NPA-005 1.0 ng0.28425 NPA-005 0.1 ng 0.27675 NPA-005 0.01 ng 0.2865

TABLE 16 HEK293, NPA-003, 24-well, n = 4 Formulation FL4 MFI Untreatedcontrol 0.3225 NPA-003 250 ng 2.9575 NPA-003 100 ng 1.255 NPA-003 10 ng0.40025 NPA-003 1 ng 0.33025 NPA-003 0.1 ng 0.34625 NPA-003 0.01 ng0.3475

TABLE 17 HEK293, NPA-003, 24-well, n = 4 Formulation FL4 MFI Untreatedcontrol 0.27425 NPA-003 250 ng 5.6075 NPA-003 100 ng 3.7825 NPA-003 30ng 1.5525

TABLE 18 Concentration and MFI MFI mCherry Formulation NPA-003 NPA-00525 ng/well 11963.25 12256.75 0.25 ng/well 1349.75 2572.75 0.025 ng/well459.50 534.75 0.0025 ng/well 310.75 471.75

Example 13 Manual Injection and Syringe Pump Formulations

Two formulations of DLin-KC2-DMA and 98N12-5 were prepared by manualinjection (MI) and syringe pump injection (SP) and analyzed along with abackground sample to compare the MFI of mCherry (mRNA sequence shown inSEQ ID NO: 21439; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Capl; fully modified with 5-methylcytosine andpseudouridine) of the different formulations. Table 19 shows that thesyringe pump formulations had a higher MFI as compared to the manualinjection formulations of the same lipid and lipid/RNA ratio.

TABLE 19 Formulations and MFI Lipid/ Method Formulation RNA Mean size offormu- # Lipid wt/wt (nm) lation MFI Untreated N/A N/A N/A N/A 674.67Control NPA-002 DLin-KC2- 15 140 nm MI 10318.25 DMA PDI: 0.11 NPA-002-2DLin-KC2- 15 105 nm SP 37054.75 DMA PDI: 0.04 NPA-003 DLin-KC2- 20 114nm MI 22037.5 DMA PDI: 0.08 NPA-003-2 DLin-KC2- 20 95 nm SP 37868.75 DMAPDI: 0.02 NPA-005 98N12-5 15 127 nm MI 11504.75 PDI: 0.12 NPA-005-298N12-5 15 106 nm SP 9343.75 PDI: 0.07 NPA-006 98N12-5 20 126 nm MI11182.25 PDI: 0.08 NPA-006-2 98N12-5 20 93 nm SP 5167 PDI: 0.08

Example 14 Lipid Nanoparticle Formulations

Formulations of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 andDLin-MC3-DMA were incubated at a concentration of 60 ng/well or 62.5ng/well in a plate of HEK293 and 62.5 ng/well in a plate of HepG2 cellsfor 24 hours to determine the MFI of mCherry (SEQ ID NO: 21439; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) for eachformulation. The formulations tested are outlined in Table 20 below. Asshown in Table 21 for the 60 ng/well and Tables 22, 23, 24 and 25 forthe 62.5 ng/well, the formulation of NPA-003 and NPA-018 have thehighest mCherry MFI and the formulations of NPA-008, NPA-010 and NPA-013are most the similar to the background sample mCherry MFI value.

TABLE 20 Formulations Formulation Lipid/RNA # Lipid wt/wt Mean size (nm)NPA-001 DLin-KC2-DMA 10 155 nm PDI: 0.08 NPA-002 DLin-KC2-DMA 15 140 nmPDI: 0.11 NPA-002-2 DLin-KC2-DMA 15 105 nm PDI: 0.04 NPA-003DLin-KC2-DMA 20 114 nm PDI: 0.08 NPA-003-2 DLin-KC2-DMA 20 95 nm PDI:0.02 NPA-005 98N12-5 15 127 nm PDI: 0.12 NPA-006 98N12-5 20 126 nm PDI:0.08 NPA-007 DLin-DMA 15 148 nm PDI: 0.09 NPA-008 DLin-K-DMA 15 121 nmPDI: 0.08 NPA-009 C12-200 15 138 nm PDI: 0.15 NPA-010 DLin-MC3-DMA 15126 nm PDI: 0.09 NPA-012 DLin-DMA 20 86 nm PDI: 0.08 NPA-013 DLin-K-DMA20 104 nm PDI: 0.03 NPA-014 C12-200 20 101 nm PDI: 0.06 NPA-015DLin-MC3-DMA 20 109 nm PDI: 0.07

TABLE 21 HEK293, 96-well, 60 ng Modified RNA/well Formulation MFImCherry Untreated 871.81 NPA-001 6407.25 NPA-002 14995 NPA-003 29499.5NPA-005 3762 NPA-006 2676 NPA-007 9905.5 NPA-008 1648.75 NPA-009 2348.25NPA-010 4426.75 NPA-012 11466 NPA-013 2098.25 NPA-014 3194.25 NPA-01514524

TABLE 22 HEK293, 62.5 ng/well Formulation MFI mCherry Untreated 871.81NPA-001 6407.25 NPA-002 14995 NPA-003 29499.5 NPA-005 3762 NPA-006 2676NPA-007 9905.5 NPA-008 1648.75 NPA-009 2348.25 NPA-010 4426.75 NPA-01211466 NPA-013 2098.25 NPA-014 3194.25 NPA-015 14524

TABLE 23 HEK293, 62.5 ng/well Formulation MFI mCherry Untreated 295NPA-007 3504 NPA-012 8286 NPA-017 6128 NPA-003-2 17528 NPA-018 34142NPA-010 1095 NPA-015 5859 NPA-019 3229

TABLE 24 HepG2, 62.5 ng/well Formulation MFI mCherry Untreated 649.94NPA-001 6006.25 NPA-002 8705 NPA-002-2 15860.25 NPA-003 15059.25NPA-003-2 28881 NPA-005 1676 NPA-006 1473 NPA-007 15678 NPA-008 2976.25NPA-009 961.75 NPA-010 3301.75 NPA-012 18333.25 NPA-013 5853 NPA-0142257 NPA-015 16225.75

TABLE 25 HepG2, 62.5 ng/well Formulation MFI mCherry Untreated control656 NPA-007 16798 NPA-012 21993 NPA-017 20377 NPA-003-2 35651 NPA-01840154 NPA-010 2496 NPA-015 19741 NPA-019 16373

Example 15 In Vivo Formulation Studies

Rodents (n=5) are administered intravenously, subcutaneously orintramuscularly a single dose of a formulation containing a modifiedmRNA and a lipid. The modified mRNA administered to the rodents isselected from G-CSF (mRNA sequence shown in SEQ ID NO: 21438; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),erythropoietin (EPO) (mRNA sequence shown in SEQ ID NO: 1638; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),Factor IX (mRNA sequence shown in SEQ ID NO: 1622; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) ormCherry (mRNA sequence shown in SEQ ID NO: 21439; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1). Theerythropoietin cDNA with the T7 promoter, 5′ untranslated region (UTR)and 3′ UTR used in in vitro transcription (IVT) is given in SEQ ID NO:21441 and SEQ ID NO: 21442.

Each formulation also contains a lipid which is selected from one ofDLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA,reLNP, ATUPLEX®, DACC and DBTC. The rodents are injected with 100 ug, 10ug or 1 ug of the formulated modified mRNA and samples are collected atspecified time intervals.

Serum from the rodents administered formulations containing human G-CSFmodified mRNA are measured by specific G-CSF ELISA and serum from miceadministered human factor IX modified RNA is analyzed by specific factorIX ELISA or chromogenic assay. The liver and spleen from the miceadministered with mCherry modified mRNA are analyzed byimmunohistochemistry (1HC) or fluorescence-activated cell sorting(FACS). As a control, a group of mice are not injected with anyformulation and their serum and tissue are collected analyzed by ELISA,FACS and/or IHC.

A. Time Course

The rodents are administered formulations containing at least onemodified mRNA to study the time course of protein expression for theadministered formulation. The rodents are bled at specified timeintervals prior to and after administration of the modified mRNAformulations to determine protein expression and complete blood count.Samples are also collected from the site of administration of rodentsadministered modified mRNA formulations subcutaneously andintramuscularly to determine the protein expression in the tissue.

B. Dose Response

The rodents are administered formulations containing at least onemodified mRNA to determine dose response of each formulation. Therodents are bled at specified time intervals prior to and afteradministration of the modified mRNA formulations to determine proteinexpression and complete blood count. The rodents are also sacrified toanalyze the effect of the modified mRNA formulation on the internaltissue. Samples are also collected from the site of administration ofrodents administered modified mRNA formulations subcutaneously andintramuscularly to determine the protein expression in the tissue.

C. Toxicity

The rodents are administered formulations containing at least onemodified mRNA to study toxicity of each formulation. The rodents arebled at specified time intervals prior to and after administration ofthe modified mRNA formulations to determine protein expression andcomplete blood count. The rodents are also sacrificed to analyze theeffect of the modified mRNA formulation on the internal tissue. Samplesare also collected from the site of administration of rodentsadministered modified mRNA formulations subcutaneously andintramuscularly to determine the protein expression in the tissue.

Example 16 PLGA Microsphere Formulations

Optimization of parameters used in the formulation of PLGA microspheresmay allow for tunable release rates and high encapsulation efficiencieswhile maintaining the integrity of the modified RNA encapsulated in themicrospheres. Parameters such as, but not limited to, particle size,recovery rates and encapsulation efficiency may be optimized to achievethe optimal formulation.

A. Synthesis of PLGA microspheres

Polylacticglycolic acid (PLGA) microspheres were synthesized using thewater/oil/water double emulsification methods known in the art usingPLGA (Lactel, Cat# B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA),polyvinylalcohol (PVA) (Sigma, Cat#348406-25G, MW 13-23k)dichloromethane and water. Briefly, 0.1 ml of water (W1) was added to 2ml of PLGA dissolved in dichloromethane (DCM) (01) at concentrationsranging from 50-200 mg/ml of PLGA. The W1/O1 emulsion was homogenized(IKA Ultra-Turrax Homogenizer, T18) for 30 seconds at speed 4 (˜15,000rpm). The W1/O1 emulsion was then added to 100 to 200 ml of 0.3 to 1%PVA (W2) and homogenized for 1 minute at varied speeds. Formulationswere left to stir for 3 hours and then washed by centrifugation (20-25min, 4,000 rpm, 4° C.). The supernatant was discarded and the PLGApellets were resuspended in 5-10 ml of water, which was repeated 2×.Average particle size (represents 20-30 particles) for each formulationwas determined by microscopy after washing. Table 26 shows that anincrease in the PLGA concentration led to larger sized microspheres. APLGA concentration of 200 mg/mL gave an average particle size of 14.8μm, 100 mg/mL was 8.7 μm, and 50 mg/mL of PLGA gave an average particlesize of 4.0 μm.

TABLE 26 Varied PLGA Concentration PLGA Sam- O1 Concen- W2 PVA Averageple Volume tration Volume Concentration Size ID (mL) (mg/mL) (mL) (%)Speed (μm) 1 2 200 100 0.3 5 14.8 2 2 100 100 0.3 5 8.7 3 2 50 100 0.3 54.0

Table 27 shows that decreasing the homogenization speed from 5 (˜20,000rpm) to speed 4 (˜15,000 rpm) led to an increase in particle size from14.8 μm to 29.7 μm.

TABLE 27 Varied Homogenization Speed PLGA Sam- O1 Concen- W2 PVA Averageple Volume tration Volume Concentration Size ID (mL) (mg/mL) (mL) (%)Speed (μm) 1 2 200 100 0.3 5 14.8 4 2 200 100 0.3 4 29.7

Table 28 shows that increasing the W2 volume (i.e. increasing the ratioof W2:01 from 50:1 to 100:1), decreased average particle size slightly.Altering the PVA concentration from 0.3 to 1 wt % had little impact onPLGA microsphere size.

TABLE 28 Varied W2 Volume and Concentration PLGA Sam- O1 Concen- W2 PVAAverage ple Volume tration Volume Concentration Size ID (mL) (mg/mL)(mL) (%) Speed (μm) 1 2 200 100 0.3 5 14.8 5 2 200 200 0.3 5 11.7 6 2200 190 0.3 5 11.4 7 2 200 190 1.0 5 12.3

B. Encapsulation of modified mRNA

Modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 21438; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1;fully modified with 5-methylcytosine and pseudouridine) was dissolved inwater at a concentration of 2 mg/ml (W3). Three batches of PLGAmicrosphere formulations were made as described above with the followingparameters: 0.1 ml of W3 at 2 mg/ml, 1.6 ml of O1 at 200 mg/ml, 160 mlof W2 at 1%, and homogenized at a speed of 4 for the first emulsion(W3/O1) and homogenized at a speed of 5 for the second emulstion(W3/O1/W2). After washing by centrifugation, the formulations werefrozen in liquid nitrogen and then lyophilized for 3 days. To test theencapsulation efficiency of the formulations, the lyophilized materialwas deformulated in DCM for 6 hours followed by an overnight extractionin water. The modified RNA concentration in the samples was thendetermined by OD260. Encapsulation efficiency was calculated by takingthe actual amount of modified RNA and dividing by the starting amount ofmodified RNA. In the three batches tested, there was an encapsulationefficiency of 59.2, 49.8 and 61.3.

C. Integrity of modified mRNA encapsulated in PLGA microspheres

Modified Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 1622; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) wasdissolved in water at varied concentrations (W4) to vary the weightpercent loading in the formulation (mg modified RNA/mg PLGA*100) and todetermine encapsulation efficiency. The parameters in Table 29 were usedto make four different batches of PLGA microsphere formulations with ahomogenization speed of 4 for the first emulstion (W4/O1) and ahomogenization speed of 5 for the second emulsion (W4/O1/W2).

TABLE 29 Factor IX PLGA Microsphere Formulation Parameters Factor WeightW4 Factor IX IX O1 PLGA W2 PVA % Vol. Conc. Amount Vol. Conc. Vol. Conc.(wt %) ID (uL) (mg/ml) (ug) (ml) (mg/ml) (ml) (%) Loading A 100 2.0200.0 2.0 200 200 1.0 0.05 B 100 4.0 400.0 2.0 200 200 1.0 0.10 C 4002.0 800.0 2.0 200 200 1.0 0.20 D 400 4.0 1600.0 2.0 200 200 1.0 0.40

After lyophilization, PLGA microspheres were weighed out in 2 mleppendorf tubes to correspond to ˜10 ug of modified RNA. Lyophilizationwas found to not destroy the overall structure of the PLGA microspheres.To increase weight percent loading (wt %) for the PLGA microspheres,increasing amounts of modified RNA were added to the samples. PLGAmicrospheres were deformulated by adding 1.0 ml of DCM to each tube andthen shaking the samples for 6 hours. For modified RNA extraction, 0.5ml of water was added to each sample and the samples were shakenovernight before the concentration of modified RNA in the samples wasdetermined by OD260. To determine the recovery of the extractionprocess, unformulated Factor IX modified RNA (mRNA sequence shown in SEQID NO: 1622; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Capl; fully modified with 5-methylcytosine andpseudouridine) (deformulation control) was spiked into DCM and wassubjected to the deformulation process. Table 30 shows the loading andencapsulation efficiency for the samples. All encapsulation efficiencysamples were normalized to the deformulation control.

TABLE 30 Weight Percent Loading and Encapsulation Efficiency TheoreticalActual modified RNA modified RNA Encapsulation ID loading (wt %) loading(wt %) Efficiency (%) A 0.05 0.06 97.1 B 0.10 0.10 85.7 C 0.20 0.18 77.6D 0.40 0.31 68.1 Control — — 100.0

D. Release Study of Modified mRNA Encapsulated in PLGA Microspheres

PLGA microspheres formulated with Factor IX modified RNA (mRNA sequenceshown in SEQ ID NO: 1622; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) were deformulated as described aboveand the integrity of the extracted modified RNA was determined byautomated electrophoresis (Bio-Rad Experion). The extracted modifiedmRNA was compared against unformulated modified mRNA and thedeformulation control in order to test the integrity of the encapsulatedmodified mRNA. As shown in FIG. 4, the majority of modRNA was intact forbatch ID A, B, C and D, for the deformulated control (Deform control)and the unformulated control (Unform control).

E. Protein Expression of Modified mRNA Encapsulated in PLGA Microspheres

PLGA microspheres formulated with Factor IX modified RNA (mRNA sequenceshown in SEQ ID NO: 1622; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) were deformulated as described aboveand the protein expression of the extracted modified RNA was determinedby an in vitro transfection assay. HEK293 cells were reverse transfectedwith 250 ng of Factor IX modified RNA complexed with RNAiMAX(Invitrogen) in triplicate.

Factor IX modified RNA was diluted in nuclease-free water to aconcentration of 25 ng/μl and RNAiMAX was diluted 13.3× in serum-freeEMEM. Equal volumes of diluted modified RNA and diluted RNAiMAX weremixed together and were allowed to stand for 20 to 30 minutes at roomtemperature. Subsequently, 20 μl of the transfection mix containing 250ng of Factor IX modified RNA was added to 80 μl of a cell suspensioncontaining 30,000 cells. Cells were then incubated for 16 h in ahumidified 37° C./5% CO2 cell culture incubator before harvesting thecell culture supernatant. Factor IX protein expression in the cellsupernatant was analyzed by an ELISA kit specific for Factor IX(Molecular Innovations, Cat # HFIXKT-TOT) and the protein expression isshown in Table 31 and FIG. 5. In all PLGA microsphere batches tested,Factor IX modified RNA remained active and expressed Factor IX proteinafter formulation in PLGA microspheres and subsequent deformulation.

TABLE 31 Protein Expression Factor IX Protein Sample Expression (ng/ml)Batch A 0.83 Batch B 1.83 Batch C 1.54 Batch D 2.52 Deformulated Control4.34 Unformulated Control 3.35

F. Release study of modified mRNA encapsulated in PLGA microspheres

PLGA micropsheres formulated with Factor IX modified RNA (mRNA sequenceshown in SEQ ID NO: 1622; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Capl; fully modified with5-methylcytosine and pseudouridine) were resuspended in water to a PLGAmicrosphere concentration of 24 mg/ml. After resuspension, 150 ul of thePLGA microsphere suspension was aliquoted into eppendorf tubes. Sampleswere kept incubating and shaking at 37° C. during the course of thestudy. Triplicate samples were pulled at 0.2, 1, 2, 8, 14, and 21 days.To determine the amount of modified RNA released from the PLGAmicrospheres, samples were centrifuged, the supernatant was removed, andthe modified RNA concentration in the supernatant was determined by OD260. The percent release, shown in Table 32, was calculated based on thetotal amount of modified RNA in each sample. After 31 days, 96% of theFactor IX modified RNA was released from the PLGA microsphereformulations.

TABLE 32 Percent Release Time (days) % Release 0 0.0 0.2 27.0 1 37.7 245.3 4 50.9 8 57.0 14 61.8 21 75.5 31 96.4

G. Particle Size Reproducibility of PLGA Microspheres

Three batches of Factor IX modified RNA (mRNA sequence shown in SEQ IDNO: 1622 polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Capl; fully modified with 5-methylcytosine andpseudouridine) PLGA microspheres were made using the same conditionsdescribed for Batch D, shown in Table 29, (0.4 ml of W4 at 4 mg/ml, 2.0ml of O1 at 200 mg/ml, 200 ml of W2 at 1%, and homogenized at a speed of5 for the W4/O1/W2 emulsion). To improve the homogeneity of the PLGAmicrosphere suspension, filtration was incorporated prior tocentrifugation. After stirring for 3 hours and before centrifuging, allformulated material was passed through a 100 μm nylon mesh strainer(Fisherbrand Cell Strainer, Cat #22-363-549) to remove largeraggregates. After washing and resuspension with water, 100-200 μl of aPLGA microspheres sample was used to measure particle size of theformulations by laser diffraction (Malvern Mastersizer2000). Theparticle size of the samples is shown in Table 33.

TABLE 33 Particle Size Summary Volume Weighted ID D10 (μm) D50 (μm) D90(μm) Mean (um) Filtration Control 19.2 62.5 722.4 223.1 No A 9.8 31.665.5 35.2 Yes B 10.5 32.3 66.9 36.1 Yes C 10.8 35.7 79.8 41.4 Yes

Results of the 3 PLGA microsphere batches using filtration were comparedto a PLGA microsphere batch made under the same conditions withoutfiltration. The inclusion of a filtration step before washing reducedthe mean particle size and demonstrated a consistent particle sizedistribution between 3 PLGA microsphere batches.

H. Serum Stability of Factor IX PLGA Microspheres

Factor IX mRNA RNA (mRNA sequence shown in SEQ ID NO: 1622 polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Capl; fullymodified with 5-methylcytosine and pseudouridine) in buffer (TE) or 90%serum (Se), or Factor IX mRNA in PLGA in buffer, 90% serum or 1% serumwas incubated in buffer, 90% serum or 1% serum at an mRNA concentrationof 50 ng/ul in a total volume of 70 ul. The samples were removed at 0,30, 60 or 120 minutes. RNases were inactivated with proteinase Kdigestion for 20 minutes at 55° C. by adding 25 ul of 4× proteinase Kbuffer (0.4 ml 1M TRIS-HCl pH 7.5, 0.1 ml 0.5M EDTA, 0.12 ml 5M NaCl,and 0.4 m1 10% SDS) and 8 ul of proteinase K at 20 mg/ml. The Factor IXmRNA was precipitated (add 250 ul 95% ethanol for 1 hour, centrifuge for10 min at 13 k rpm and remove supernatant, add 200 ul 70% ethanol to thepellet, centrifuge again for 5 min at 13 k rpm and remove supernatantand resuspend the pellet in 70 ul water) or extracted from PLGAmicrospheres (centrifuge 5 min at 13k rpm and remove supernatant, washpellet with 1 ml water, centrifuge 5 min at 13k rpm and removesupernatant, add 280 ul dichloromethane to the pellet and shake for 15minutes, add 70 ul water and then shake for 2 hours and remove theaqueous phase) before being analyzed by bioanalyzer. PLGA microspheresprotect Factor IX modified mRNA from degradation in 90% and 1% serumover 2 hours. Factor IX modified mRNA completely degrades in 90% serumat the initial time point.

Example 17 Lipid Nanoparticle In Vivo Studies

G-CSF (cDNA with the T7 promoter, 5′ Untranslated region (UTR) and 3′UTRused in in vitro transcription is given in SEQ ID NO: 21437. mRNAsequence shown in SEQ ID NO: 21438; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap 1; fully modified with5-methylcytosine and pseudouridine) and Factor IX (cDNA with the T7promoter, 5′ UTR and 3′UTR used in vitro transcription is given in SEQID NO: 21443. mRNA sequence shown in SEQ ID NO: 1622; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap 1;fully modified with 5-methylcytosine and pseudouridine) modified mRNAwere formulated as lipid nanoparticles (LNPs) using the syringe pumpmethod. The LNPs were formulated at a 20:1 weight ratio of total lipidto modified mRNA with a final lipid molar ratio of 50:10:38.5:1.5(DLin-KC2-DMA: DSPC: Cholesterol: PEG-c-DOMG). Formulations, listed inTable 34, were characterized by particle size, zeta potential, andencapsulation.

TABLE 34 Formulations Formulation # NPA-029-1 NPA-030-1 Modified mRNAFactor IX G-CSF Mean size 91 nm 106 nm PDI: 0.04 PDI: 0.06 Zeta at pH7.4 1.8 mV 0.9 mV Encaps. 92% 100% (RiboGr)

LNP formulations were administered to mice (n=5) intravenously at amodified mRNA dose of 100, 10, or 1 ug. Mice were sacrificed at 8 hrsafter dosing. Serum was collected by cardiac puncture from mice thatwere administered with G-CSF or Factor IX modified mRNA formulations.Protein expression was determined by ELISA.

There was no significant body weight loss (<5%) in the G-CSF or FactorIX dose groups. Protein expression for G-CSF or Factor IX dose groupswas determined by ELISA from a standard curve. Serum samples werediluted (about 20-2500× for G-CSF and about 10-250× for Factor IX) toensure samples were within the linear range of the standard curve. Asshown in Table 35, G-CSF protein expression determined by ELISA wasapproximately 17, 1200, and 4700 ng/ml for the 1, 10, and 100 ug dosegroups, respectively. As shown in Table 36, Factor IX protein expressiondetermined by ELISA was approximately 36, 380, and 3000-11000 ng/ml forthe 1, 10, and 100 ug dose groups, respectively.

TABLE 35 G-CSF Protein Expression Dose (ug) Conc (ng/ml) Dilution FactorSample Volume 1 17.73  20x   5 ul 10 1204.82 2500x 0.04 ul 100 4722.202500x 0.04 ul

TABLE 36 Factor IX Protein Expression Dose (ug) Conc (ng/ml) DilutionFactor Sample Volume  1 36.05 10x 5 ul 10 383.04 10x 5 ul 100* 3247.7550x 1 ul 100* 11177.20 250x  0.2 ul  

As shown in Table 37, the LNP formulations described above have about a10,000-100.000-fold increase in protein production compared to anadministration of an intravenous (IV)-lipoplex formulation for the samedosage of modified mRNA and intramuscular (IM) or subcutaneous (SC)administration of the same dose of modified mRNA in saline. As used inTable 37, the symbol “˜” means about.

TABLE 37 Protein Production Serum Concentration (pg/ml) G-CSF Dose (ug)8-12 hours after administration IM 100 ~20-80 SC 100 ~10-40 IV(Lipoplex) 100 ~30 IV (LNP) 100 ~5,000,000 IV (LNP) 10 ~1,000,000 IV(LNP) 1 ~20,000 Serum Concentration (ng/ml) Factor IX Dose (ug) 8-12hours after administration IM 2 x 100 ~1.6 ng/ml IV (LNP) 100~3,000-10,000 ng/ml IV (LNP) 10 ~400 ng/ml IV (LNP) 1 ~40 ng/ml

Materials and Methods for Examples 18-23

G-CSF (mRNA sequence shown in SEQ ID NO: 21438; polyA tail ofapproximately 160 nulceotides not shown in sequence; 5′ cap, Cap 1;fully modified with 5-methylcytosine and pseudouridine) and EPO (mRNAsequence shown in SEQ ID NO: 1638; polyA tail of approximately 160nulceotides not shown in sequence; 5′ cap, Cap 1; fully modified with5-methylcytosine and pseudouridine) modified mRNA were formulated aslipid nanoparticles (LNPs) using the syringe pump method. The LNPs wereformulated at a 20:1 weight ratio of total lipid to modified mRNA with afinal lipid molar ratio of 50:10:38.5:1.5 (DLin-KC2-DMA: DSPC:Cholesterol: PEG-c-DOMG). Formulations, listed in Table 38, werecharacterized by particle size, zeta potential, and encapsulation.

TABLE 38 Formulations Formulation # NPA-030-2 NPA-060-1 Modified mRNAG-CSF EPO Mean size 84 nm 85 nm PDI: 0.04 PDI: 0.03 Zeta at pH 7.4 0.8mV 1.5 mV Encapsulation 95% 98% (RiboGreen)

Example 18 Lipid Nanoparticle In Vivo Studies with Modified mRNA

LNP formulations, shown in Table 38 (above), were administered to rats(n=5) intravenously (IV), intramuscularly (IM) or subcutaneously (SC) ata single modified mRNA dose of 0.05 mg/kg. A control group of rats (n=4)was untreated. The rats were bled at 2 hours, 8 hours, 24 hours, 48hours and 96 hours and after they were administered with G-CSF or EPOmodified mRNA formulations to determine protein expression using ELISA.The rats administered EPO modified mRNA intravenously were also bled at7 days.

As shown in Table 39, EPO protein expression in the rats intravenouslyadministered modified EPO mRNA was detectable out to 5 days. G-CSF inthe rats intravenously administered modified G-CSF mRNA was detectableto 7 days. Subcutaneous and intramuscular administration of EPO modifiedmRNA was detectable to at least 24 hours and G-CSF modified mRNA wasdetectable to at least 8 hours. In

Table 39, “OSC” refers to values that were outside the standard curveand “NT” means not tested.

TABLE 39 G-CSF and EPO Protein Expression EPO Serum G-CSF Serum RouteTime Concentration (pg/ml) Concentration (pg/ml) IV  2 hours 36,981.031,331.9 IV  8 hours 62,053.3 70,532.4 IV 24 hours 42,077.0 5,738.6 IV48 hours 5,561.5 233.8 IV  5 days 0.0 60.4 IV  7 days 0.0 NT IM  2 hours1395.4 1620.4 IM  8 hours 8974.6 7910.4 IM 24 hours 4678.3 893.3 IM 48hours NT OSC IM  5 days NT OSC SC  2 hours 386.2 80.3 SC  8 hours 985.6164.2 SC 24 hours 544.2 OSC SC 48 hours NT OSC SC  5 days NT OSCUntreated All bleeds 0 0

Example 19 Time Course In Vivo Study

LNP formulations, shown in Table 38 (above), were administered to mice(n=5) intravenously (IV) at a single modified mRNA dose of 0.5, 0.05 or0.005 mg/kg. The mice were bled at 8 hours, 24 hours, 72 hours and 6days after they were administered with G-CSF or EPO modified mRNAformulations to determine protein expression using ELISA.

As shown in Table 40, EPO and G-CSF protein expression in the miceadministered with the modified mRNA intravenously was detectable out to72 hours for the mice dosed with 0.005 mg/kg and 0.05 mg/kg of modifiedmRNA and out to 6 days for the mice administered the EPO modified mRNA.In Table 40, “>” means greater than and “ND” means not detected.

TABLE 40 Protein Expression EPO Serum G-CSF Serum ConcentrationConcentration Dose (mg/kg) Time (pg/ml) (pg/ml) 0.005  8 hours 12,508.311,550.6 0.005 24 hours 6,803.0 5,068.9 0.005 72 hours ND ND 0.005  6days ND ND 0.05  8 hours 92,139.9 462,312.5 0.05 24 hours 54,389.480,903.8 0.05 72 hours ND ND 0.05  6 days ND ND 0.5  8 hours498,515.3 >1,250,000 0.5 24 hours 160,566.3 495,812.5 0.5 72 hours3,492.5 1,325.6 0.5  6 days 21.2 ND

Example 20 LNP Formulations In Vivo Study in Rodents

A. LNP Formulations in Mice

LNP formulations, shown in Table 38 (above), were administered to mice(n=4) intravenously (IV) at a single modified mRNA dose 0.05 mg/kg or0.005 mg/kg. There was also 3 control groups of mice (n=4) that wereuntreated. The mice were bled at 2 hours, 8 hours, 24 hours, 48 hoursand 72 hours after they were administered with G-CSF or EPO modifiedmRNA formulations to determine the protein expression. Proteinexpression of G-CSF and EPO were determined using ELISA.

As shown in Table 41, EPO and G-CSF protein expression in the mice wasdetectable at least out to 48 hours for the mice that received a dose of0.005 mg/kg modified RNA and 72 hours for the mice that received a doseof 0.05 mg/kg modified RNA. In Table 41, “OSC” refers to values thatwere outside the standard curve and “NT” means not tested.

TABLE 41 Protein Expression in Mice EPO Serum G-CSF Serum ConcentrationConcentration Dose (mg/kg) Time (pg/ml) (pg/ml) 0.005  2 hours OSC3,447.8 0.005  8 hours 1,632.8 11,454.0 0.005 24 hours 1,141.0 4,960.20.005 48 hours 137.4 686.4 0.005 72 hours 0 NT 0.05  2 hours 10,027.320,951.4 0.05  8 hours 56,547.2 70,012.8 0.05 24 hours 25,027.3 19,356.20.05 48 hours 1,432.3 1,963.0 0.05 72 hours 82.2 47.3

B. LNP Formulations in Rodents

LNP formulations, shown in Table 38 (above), are administered to rats(n=4) intravenously (IV) at a single modified mRNA dose 0.05 mg/kg.There is also a control group of rats (n=4) that are untreated. The ratsare bled at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours, 7 days and14 days after they were administered with G-CSF or EPO modified mRNAformulations to determine the protein expression. Protein expression ofG-CSF and EPO are determined using ELISA.

Example 21 Early Time Course Study of LNPs

LNP formulations, shown in Table 38 (above), are administered to mammalsintravenously (IV), intramuscularly (IM) or subcutaneously (SC) at asingle modified mRNA dose of 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg. Acontrol group of mammals are not treated. The mammals are bled at 5minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5hours and/or 2 hours after they are administered with the modified mRNALNP formulations to determine protein expression using ELISA. Themammals are also bled to determine the complete blood count such as thegranulocyte levels and red blood cell count.

Example 22 Non-Human Primate In Vivo Study

LNP formulations, shown in Table 38 (above), were administered tonon-human primates (NHP) (cynomolgus monkey) (n=2) as a bolusintravenous injection (IV) over approximately 30 seconds using ahypodermic needle, which may be attached to a syringe/abbocath orbutterfly if needed. The NHP were administered a single modified mRNA IVdose of 0.05 mg/kg of EPO or G-CSF or 0.005 mg/kg of EPO in a dosevolume of 0.5 mL/kg. The NHPs were bled 5-6 days before dosing with themodified mRNA LNP formulations to determine protein expression in theserum and a baseline complete blood count. After administration with themodified mRNA formulation the NHP were bled at 8, 24, 48 and 72 hours todetermined protein expression. At 24 and 72 hours after administrationthe complete blood count of the NHP was also determined. Proteinexpression of G-CSF and EPO was determined by ELISA. Urine from the NHPswas collected over the course of the entire experiment and analyzed toevaluate clinical safety. Samples were collected from the NHPs afterthey were administered with G-CSF or EPO modified mRNA formulations todetermine protein expression using ELISA. Clinical chemistry,hematology, urinalysis and cytokines of the non-human primates were alsoanalyzed.

As shown in Table 42, EPO protein expression in the NHPs administered0.05 mg/kg is detectable out to 72 hours and the 0.005 mg/kg dosing ofthe EPO formulation is detectable out to 48 hours. In Table 42, the “<”means less than a given value. G-CSF protein expression was seen out to24 hours after administration with the modified mRNA formulation.Preliminarily, there was an increase in granulocytes and reticulocyteslevels seen in the NHP after administration with the modified mRNAformulations.

TABLE 42 Protein Expression in Non-Human Primates Female NHP Male NHPAverage Serum Serum Serum Concen- Concen- Conen- Modified Dose trationtration tration mRNA (mg/kg) Time (pg/ml) (pg/ml) (pg/ml) G-CSF 0.05Pre-bleed 0 0 0  8 hours 3289 1722 2,506 24 hours 722 307 515 48 hours 00 0 72 hours 0 0 0 EPO 0.05 Pre-bleed 0 0 0  8 hours 19,858 7,072 13,46524 hours 18,178 4,913 11,546 48 hours 5,291 498 2,895 72 hours 744 60402 EPO 0.005 Pre-bleed 0 0 0  8 hours 523 250 387 24 hours 302 113 20848 hours <7.8 <7.8 <7.8 72 hours 0 0 0

Example 23 Non-Human Primate In Vivo Study for G-CSF and EPO

LNP formulations, shown in Table 38 (above), were administered tonon-human primates (NHP) (cynomolgus monkey) (n=2) as intravenousinjection (IV). The NHP were administered a single modified mRNA IV doseof 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg of G-CSF or EPO in a dose volumeof 0.5 mL/kg. The NHPs were bled before dosing with the modified mRNALNP formulations to determine protein expression in the serum and abaseline complete blood count. After administration with the G-CSFmodified mRNA formulation the NHP were bled at 8, 24, 48 and 72 hours todetermined protein expression. After administration with the EPOmodified mRNA formulation the NHP were bled at 8, 24, 48, 72 hours and 7days to determined protein expression.

Samples collected from the NHPs after they were administered with G-CSFor EPO modified mRNA formulations were analyzed by ELISA to determineprotein expression. Neutrophil and reticulocyte count was alsodetermined pre-dose, 24 hours, 3 days, 7 days, 14 days and 18 days afteradministration of the modified G-CSF or EPO formulation.

As shown in Table 43, G-CSF protein expression was not detected beyond72 hours. In Table 43, “<39” refers to a value below the lower limit ofdetection of 39 pg/ml.

TABLE 43 G-CSF Protein Expression Female NHP Male NHP Serum G-CSF SerumG-CSF Modified Dose Concentration Concentration mRNA (mg/kg) Time(pg/ml) (pg/ml) G-CSF 0.5 Pre-bleed <39 <39  8 hours 43,525 43,594 24hours 11,374 3,628 48 hours 1,100 833 72 hours <39 306 G-CSF 0.05Pre-bleed <39 <39  8 hours 3,289 1,722 24 hours 722 307 48 hours <39 <3972 hours <39 <39 G-CSF 0.005 Pre-bleed <39 <39  8 hours 559 700 24 hours155 <39 48 hours <39 <39 72 hours <39 <39

As shown in Table 44, EPO protein expression was not detected beyond 7days. In Table 44, “<7.8” refers to a value below the lower limit ofdetection of 7.8 pg/ml.

TABLE 44 EPO Protein Expression Female NHP Male NHP Serum EPO Serum EPOModified Dose Concentration Concentration mRNA (mg/kg) Time (pg/ml)(pg/ml) EPO 0.5 Pre-bleed <7.8 <7.8  8 hours 158,771 119,086 24 hours133,978 85,825 48 hours 45,250 64,793 72 hours 15,097 20,407  7 days<7.8 <7.8 EPO 0.05 Pre-bleed <7.8 <7.8  8 hours 19,858 7,072 24 hours18,187 4,913 48 hours 5,291 498 72 hours 744 60  7 days <7.8 <7.8 EPO0.005 Pre-bleed <7.8 <7.8  8 hours 523 250 24 hours 302 113 48 hours 1129 72 hours <7.8 <7.8  7 days <7.8 <7.8

As shown in Table 45, there was an increase in neutrophils in all G-CSFgroups relative to pre-dose levels.

TABLE 45 Pharmacologic Effect of G-CSF mRNA in NHP Male NHP Female NHPMale NHP Female NHP (G-CSF) (G-CSF) (EPO) (EPO) Dose NeutrophilsNeutrophils Neutrophils Neutrophils (mg/kg) Time (10⁹/L) (10⁹/L) (10⁹/L)(10⁹/L) 0.5 Pre-dose 1.53 1.27 9.72 1.82 24 hours  14.92 13.96 7.5 11.853 days 9.76 13.7 11.07 5.22 7 days 2.74 3.81 11.8 2.85 14/18 days   2.58 1.98 7.16 2.36 0.05 Pre-dose 13.74 3.05 0.97 2.15 24 hours  19.9229.91 2.51 2.63 3 days 7.49 10.77 1.73 4.08 7 days 4.13 3.8 1.23 2.7714/18 days    3.59 1.82 1.53 1.27 0.005 Pre-dose 1.52 2.54 5.46 5.96 24hours  16.44 8.6 5.37 2.59 3 days 3.74 1.78 6.08 2.83 7 days 7.28 2.273.51 2.23 14/18 days    4.31 2.28 1.52 2.54

As shown in Table 46, there was an increase in reticulocytes in all EPOgroups 3 days to 14/18 days after dosing relative to reticulocyte levels24 hours after dosing.

TABLE 46 Pharmacologic Effect of EPO mRNA on Neutrophil Count Male NHPFemale NHP Male NHP Female NHP (G-CSF) (G-CSF) (EPO) (EPO) DoseNeutrophils Neutrophils Neutrophils Neutrophils (mg/kg) Time (10¹²/L)(10¹²/L) (10¹²/L) (10¹²/L) 0.5 Pre-dose 0.067 0.055 0.107 0.06 24 hours 0.032 0.046 0.049 0.045 3 days 0.041 0.017 0.09 0.064 7 days 0.009 0.0210.35 0.367 14/18 days    0.029 0.071 0.066 0.071 0.05 Pre-dose 0.0550.049 0.054 0.032 24 hours  0.048 0.046 0.071 0.04 3 days 0.101 0.0610.102 0.105 7 days 0.157 0.094 0.15 0.241 14/18 days    0.107 0.06 0.0670.055 0.005 Pre-dose 0.037 0.06 0.036 0.052 24 hours  0.037 0.07 0.0340.061 3 days 0.037 0.054 0.079 0.118 7 days 0.046 0.066 0.049 0.08714/18 days    0.069 0.057 0.037 0.06

As shown in Tables 47-49, the administration of EPO modified RNA had aneffect on other erythropoetic parameters including hemoglobin (HGB),hematocrit (HCT) and red blood cell (RBC) count.

TABLE 47 Pharmacologic Effect of EPO mRNA on Hemoglobin Dose Male NHP(G- Female NHP (G- Male NHP (EPO) Female NHP (EPO) (mg/kg) Time CSF) HGB(g/L) CSF) HGB (g/L) HGB (g/L) HGB (g/L) 0.5 Pre-dose 133 129 134 123 24hours  113 112 127 108 3 days 118 114 126 120 7 days 115 116 140 13414/18 days    98 113 146 133 0.05 Pre-dose 137 129 133 133 24 hours  122117 123 116 3 days 126 115 116 120 7 days 126 116 126 121 14/18 days   134 123 133 129 0.005 Pre-dose 128 129 132 136 24 hours  117 127 122 1283 days 116 127 125 130 7 days 116 129 119 127 14/18 days    118 129 128129

TABLE 48 Pharmacologic Effect of EPO mRNA on Hematocrit Dose Male NHP(G- Female NHP (G- Male NHP (EPO) Female NHP (EPO) (mg/kg) Time CSF) HCT(L/L) CSF) HCT (L/L) HCT (L/L) HCT (L/L) 0.5 Pre-dose 0.46 0.43 0.44 0.424 hours  0.37 0.38 0.4 0.36 3 days 0.39 0.38 0.41 0.39 7 days 0.39 0.380.45 0.45 14/18 days    0.34 0.37 0.48 0.46 0.05 Pre-dose 0.44 0.44 0.450.43 24 hours  0.39 0.4 0.43 0.39 3 days 0.41 0.39 0.38 0.4 7 days 0.420.4 0.45 0.41 14/18 days    0.44 0.4 0.46 0.43 0.005 Pre-dose 0.42 0.420.48 0.45 24 hours  0.4 0.42 0.42 0.43 3 days 0.4 0.41 0.44 0.42 7 days0.39 0.42 0.41 0.42 14/18 days    0.41 0.42 0.42 0.42

TABLE 49 Pharmacologic Effect of EPO mRNA on Red Blood Cells Male NHP(G- Female NHP (G- Dose CSF) RBC CSF) RBC Male NHP (EPO) Female NHP(EPO) (mg/kg) Time (10¹²/L) (10¹²/L) RBC (10¹²/L) RBC (10¹²/L) 0.5Pre-dose 5.57 5.57 5.43 5.26 24 hours  4.66 4.96 5.12 4.69 3 days 4.914.97 5.13 5.15 7 days 4.8 5.04 5.55 5.68 14/18 days    4.21 4.92 5.835.72 0.05 Pre-dose 5.68 5.64 5.57 5.84 24 hours  4.96 5.08 5.25 5.18 3days 5.13 5.04 4.81 5.16 7 days 5.17 5.05 5.37 5.31 14/18 days    5.435.26 5.57 5.57 0.005 Pre-dose 5.67 5.36 6.15 5.72 24 hours  5.34 5.355.63 5.35 3 days 5.32 5.24 5.77 5.42 7 days 5.25 5.34 5.49 5.35 14/18days    5.37 5.34 5.67 5.36

As shown in Tables 50 and 51, the administration of modified RNA had aneffect on serum chemistry parameters including alanine transaminase(ALT) and aspartate transaminase (AST).

TABLE 50 Pharmacologic Effect of EPO mRNA on Alanine Transaminase DoseMale NHP (G-CSF) Female NHP (G-CSF) Male NHP (EPO) Female NHP (EPO)(mg/kg) Time ALT (U/L) ALT (U/L) ALT (U/L) ALT (U/L) 0.5 Pre-dose 29 21650 31 2 days 63 209 98 77 4 days 70 98 94 87 7 days 41 149 60 59 14days  43 145 88 44 0.05 Pre-dose 58 53 56 160 2 days 82 39 95 254 4 days88 56 70 200 7 days 73 73 64 187 14 days  50 31 29 216 0.005 Pre-dose 4351 45 45 2 days 39 32 62 48 4 days 48 58 48 50 7 days 29 55 21 48 14days  44 46 43 51

TABLE 51 Pharmacologic Effect of EPO mRNA on Aspartate Transaminase MaleNHP (G-CSF) Female NHP (G-CSF) Male NHP (EPO) Female NHP (EPO) Dose(mg/kg) Time AST (U/L) AST (U/L) AST (U/L) AST (U/L) 0.5 Pre-dose 32 4759 20 2 days 196 294 125 141 4 days 67 63 71 60 7 days 53 68 56 47 14days  47 67 82 44 0.05 Pre-dose 99 33 74 58 2 days 95 34 61 80 4 days 6942 48 94 7 days 62 52 53 78 14 days  59 20 32 47 0.005 Pre-dose 35 54 3940 2 days 70 34 29 25 4 days 39 36 43 55 7 days 28 31 55 31 14 days  3920 35 54

As shown in Table 52, the administration of lipidnanoparticle-formulated modified RNA at high doses (0.5 mg/kg) caused anincrease in cytokines, interferon-alpha (IFN-alpha) after administrationof modified mRNA.

TABLE 52 Pharmacologic Effect of EPO mRNA on Alanine Transaminase MaleNHP Female NHP Male NHP Female NHP (G-CSF) (G-CSF) (EPO) (EPO) DoseIFN-alpha IFN-alpha IFN-alpha IFN-alpha (mg/kg) Time (pg/mL) (pg/mL)(pg/mL) (pg/mL) 0.5 Pre-dose 0 0 0 0 Day 1 + 503.8 529.2 16.79 217.5 8hr 4 days 0 0 0 0 0.05 Pre-dose 0 0 0 0 Day 1 + 0 0 0 0 8 hr 4 days 0 00 0 0.005 Pre-dose 0 0 0 0 Day 1 + 0 0 0 0 8 hr 4 days 0 0 0 0

Example 24 Study of Intramuscular and/or Subcutaneous Administration inNon-Human Primates

Formulations containing modified EPO mRNA (mRNA sequence shown in SEQ IDNO: 1638; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Capl; fully modified with 5-methylcytosine andpseudouridine) or G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 21438;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Capl; fully modified with 5-methylcytosine and pseudouridine) insaline were administered to non-human primates (Cynomolgus monkey) (NHP)intramuscularly (IM) or subcutaneously (SC). The single modified mRNAdose of 0.05 mg/kg or 0.005 mg/kg was in a dose volume of 0.5 mL/kg. Thenon-human primates are bled 5-6 days prior to dosing to determine serumprotein concentration and a baseline complete blood count. Afteradministration with the modified mRNA formulation the NHP are bled at 8hours, 24 hours, 48 hours, 72 hours, 7 days and 14 days to determinedprotein expression. Protein expression of G-CSF and EPO is determined byELISA. At 24 hours, 72 hours, 7 days and 14 days after administrationthe complete blood count of the NHP is also determined. Urine from theNHPs is collected over the course of the entire experiment and analyzedto evaluate clinical safety. Tissue near the injection site is alsocollected and analyzed to determine protein expression.

Example 25 Modified mRNA Trafficking

In order to determine localization and/or trafficking of the modifiedmRNA, studies may be performed as follows.

LNP formulations of siRNA and modified mRNA are formulated according tomethods known in the art and/or described herein. The LNP formulationsmay include at least one modified mRNA which may encode a protein suchas G-CSF, EPO, Factor VII, and/or any protein described herein. Theformulations may be administered locally into muscle of mammals usingintramuscular or subcutaneous injection. The dose of modified mRNA andthe size of the LNP may be varied to determine the effect on traffickingin the body of the mammal and/or to assess the impact on a biologicreaction such as, but not limited to, inflammation. The mammal may bebled at different time points to determine the expression of proteinencoded by the modified mRNA administered present in the serum and/or todetermine the complete blood count in the mammal.

For example, modified mRNA encoding Factor VII, expressed in the liverand secreted into the serum, may be administered intramuscularly and/orsubcutaneously. Coincident or prior to modified mRNA administration,siRNA is administered to knock out endogenous Factor VII. Factor VIIarising from the intramuscular and/or subcutaneous injection of modifiedmRNA is administered is measured in the blood. Also, the levels ofFactor VII is measured in the tissues near the injection site. If FactorVII is expressed in blood then there is trafficking of the modifiedmRNA. If Factor VII is expressed in tissue and not in the blood thanthere is only local expression of Factor VII.

Example 26 Formulations of Multiple Modified mRNA

LNP formulations of modified mRNA are formulated according to methodsknown in the art and/or described herein or known in the art. The LNPformulations may include at least one modified mRNA which may encode aprotein such as G-CSF, EPO, thrombopoietin and/or any protein describedherein. The at least one modified mRNA may include 1, 2, 3, 4 or 5modified mRNA molecules. The formulations containing at least onemodified mRNA may be administered intravenously, intramuscularly orsubcutaneously in a single or multiple dosing regimens. Biologicalsamples such as, but not limited to, blood and/or serum may be collectedand analyzed at different time points before and/or after administrationof the at least one modified mRNA formulation. An expression of aprotein in a biological sample of 50-200 pg/ml after the mammal has beenadministered a formulation containing at least one modified mRNAencoding said protein would be considered biologically effective.

Example 27 Polyethylene Glycol Ratio Studies

A. Formulation and Characterization of PEG LNPs

Lipid nanoparticles (LNPs) were formulated using the syringe pumpmethod. The LNPs were formulated at a 20:1 weight ratio of total lipidto modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 21438; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine). The molarratio ranges of the formulations are shown in Table 53.

TABLE 53 Molar Ratios DLin- PEG-c- KC2-DMA DSPC Cholesterol DOMG MolePercent 50.0 10.0 37-38.5 1.5-3 (mol %)

Two types of PEG lipid, 1,2-Dimyristoyl-sn-glycerol, methoxypolyethyleneGlycol (PEG-DMG, NOF Cat # SUNBRIGHT® GM-020) and1,2-Distearoyl-sn-glycerol, methoxypolyethylene Glycol (PEG-DSG, NOF Cat# SUNBRIGHT® GS-020), were tested at 1.5 or 3.0 mol %. After theformation of the LNPs and the encapsulation of the modified G-CSF mRNA,the LNP formulations were characterized by particle size, zeta potentialand encapsulation percentage and the results are shown in Table 54.

TABLE 54 Characterization of LNP Formulations Formulation No. NPA-071-1NPA-072-1 NPA-073-1 NPA-074-1 Lipid PEG-DMG PEG-DMG PEG-DSA PEG-DSA 1.5%3% 1.5% 3% Mean Size 95 nm 85 nm 95 nm 75 nm PDI: 0.01 PDI: 0.06 PDI:0.08 PDI: 0.08 Zeta at pH 7.4 −1.1 mV −2.6 mV 1.7 mV 0.7 mVEncapsulation 88% 89% 98% 95% (RiboGreen)

B. In Vivo Screening of PEG LNPs

Formulations of the PEG LNPs described in Table 55 were administered tomice (n=5) intravenously at a dose of 0.5 mg/kg. Serum was collectedfrom the mice at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours and 8days after administration of the formulation. The serum was analyzed byELISA to determine the protein expression of G-CSF and the expressionlevels are shown in Table 55. LNP formulations using PEG-DMG gavesubstantially higher levels of protein expression than LNP formulationswith PEG-DSA.

TABLE 55 Protein Expression Formulation Protein Expression Lipid No.Time (pg/ml) PEG-DMG, 1.5% NPA-071-1  2 hours 114,102  8 hours 357,94424 hours 104,832 48 hours 6,697 72 hours 980  8 days 0 PEG-DMG, 3%NPA-072-1  2 hours 154,079  8 hours 354,994 24 hours 164,311 48 hours13,048 72 hours 1,182  8 days 13 PEG-DSA, 1.5% NPA-073-1  2 hours 3,193 8 hours 6,162 24 hours 446 48 hours 197 72 hours 124  8 days 5 PEG-DSA,3% NPA-074-1  2 hours 259  8 hours 567 24 hours 258 48 hours 160 72hours 328  8 days 33

Example 28 Cationic Lipid Formulation Studies

A. Formulation and Characterization of Cationic Lipid Nanoparticles

Lipid nanoparticles (LNPs) were formulated using the syringe pumpmethod.

The LNPs were formulated at a 20:1 weight ratio of total lipid tomodified mRNA. The final lipid molar ratio ranges of cationic lipid,DSPC, cholesterol and PEG-c-DOMG are outlined in Table 56.

TABLE 56 Molar Ratios Cationic PEG-c- Lipid DSPC Cholesterol DOMG MolePercent 50.0 10.0 38.5 1.5 (mol %)

A 25 mM lipid solution in ethanol and modified RNA in 50 mM citrate at apH of 3 were mixed to create spontaneous vesicle formation. The vesicleswere stabilized in ethanol before the ethanol was removed and there wasa buffer exchange by dialysis. The LNPs were then characterized byparticle size, zeta potential, and encapsulation percentage. Table 57describes the characterization of LNPs encapsulating EPO modified mRNA(mRNA sequence shown in SEQ ID NO: 1638 polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Capl; fully modified with5-methylcytosine and pseudouridine) or G-CSF modified mRNA (mRNAsequence shown in SEQ ID NO: 21438; polyA tail of approximately 160nucleotidies not shown in sequence; 5′ cap, Capl; fully modified with5-methylcytosine and pseudouridine) using DLin-MC3-DMA, DLin-DMA orC12-200 as the cationic lipid.

TABLE 57 Characterization of Cationic Lipid Formulations Formulation No.NPA- NPA- NPA-071-1 NPA-072-1 NPA-073-1 074-1 NPA-075-1 076-1 LipidDLin- DLin- DLin- DLin- C12-200 C12- MC3- MC3- DMA DMA 200 DMA DMAModified RNA EPO G-CSF EPO G-CSF EPO G-CSF Mean Size 89 nm 96 nm 70 nm73 nm 97 nm 103 nm PDI: 0.07 PDI: 0.08 PDI: 0.04 PDI: PDI: 0.05 PDI:0.06 0.09 Zeta at pH 7.4 −1.1 mV −1.4 mV −1.6 mV −0.4 mV 1.4 mV 0.9 mVEncapsulation 100% 100% 99% 100% 88% 98% (RiboGreen)

B. In Vivo Screening of Cationic LNP Formulations

Formulations of the cationic lipid formulations described in Table 57were administered to mice (n=5) intravenously at a dose of 0.5 mg/kg.Serum was collected from the mice at 2 hours, 24 hours, 72 hours and/or7 days after administration of the formulation. The serum was analyzedby ELISA to determine the protein expression of EPO or G-CSF and theexpression levels are shown in Table 58.

TABLE 58 Protein Expression Protein Modified Formulation Expression mRNANo. Time (pg/ml) EPO NPA-071-1  2 hours 304,190.0 24 hours 166,811.5 72hours 1,356.1  7 days 20.3 EPO NPA-073-1  2 hours 73,852.0 24 hours75,559.7 72 hours 130.8 EPO NPA-075-1  2 hours 413,010.2 24 hours56,463.8 G-CSF NPA-072-1  2 hours 62,113.1 24 hours 53,206.6 G-CSFNPA-074-1 24 hours 25,059.3 G-CSF NPA-076-1  2 hours 219,198.1 24 hours8,470.0

Toxcity was seen in the mice administered the LNPs formulations with thecationic lipid C12-200 (NPA-075-1 and NPA-076-1) and they weresacrificed at 24 hours because they showed symptoms such as scrubby fur,cowering behavior and weight loss of greater than 10%. C12-200 wasexpected to be more toxic but also had a high level of expression over ashort period. The cationic lipid DLin-DMA (NPA-073-1 and NPA-074-1) hadthe lowest expression out of the three cationic lipids tested.DLin-MC3-DMA (NPA-071-1 and NPA-072-1) showed good expression up to daythree and was above the background sample out to day 7 for EPOformulations.

Example 29 Method of Screening for Protein Expression

A. Electrospray Ionization

A biological sample which may contain proteins encoded by modified RNAadministered to the subject is prepared and analyzed according to themanufacturer protocol for electrospray ionization (ESI) using 1, 2, 3 or4 mass analyzers. A biologic sample may also be analyzed using a tandemESI mass spectrometry system.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

B. Matrix-Assisted Laser Desorption/Ionization

A biological sample which may contain proteins encoded by modified RNAadministered to the subject is prepared and analyzed according to themanufacturer protocol for matrix-assisted laser desorption/ionization(MALDI).

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry

A biological sample, which may contain proteins encoded by modified RNA,may be treated with a trypsin enzyme to digest the proteins containedwithin. The resulting peptides are analyzed by liquidchromatography-mass spectrometry-mass spectrometry (LC/MS/MS). Thepeptides are fragmented in the mass spectrometer to yield diagnosticpatterns that can be matched to protein sequence databases via computeralgorithms. The digested sample may be diluted to achieve 1 ng or lessstarting material for a given protein. Biological samples containing asimple buffer background (e.g. water or volatile salts) are amenable todirect in-solution digest; more complex backgrounds (e.g. detergent,non-volatile salts, glycerol) require an additional clean-up step tofacilitate the sample analysis.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

Example 30 Lipid Nanoparticle In Vivo Studies

mCherry mRNA (mRNA sequence shown in SEQ ID NO: 21444; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Capl; fullymodified with 5-methylcytosine and pseudouridine) was formulated as alipid nanoparticle (LNP) using the syringe pump method. The LNP wasformulated at a 20:1 weight ratio of total lipid to modified mRNA with afinal lipid molar ratio of 50:10:38.5:1.5 (DLin-KC2-DMA: DSPC:Cholesterol: PEG-c-DOMG). The mCherry formulation, listed in Table 59,was characterized by particle size, zeta potential, and encapsulation.

TABLE 59 mCherry Formulation Formulation # NPA-003-5 Modified mRNAmCherry Mean size 105 nm PDI: 0.09 Zeta at pH 7.4 1.8 mV Encaps.(RiboGr) 100%

The LNP formulation was administered to mice (n=5) intravenously at amodified mRNA dose of 100 ug. Mice were sacrificed at 24 hrs afterdosing. The liver and spleen from the mice administered with mCherrymodified mRNA formulations were analyzed by immunohistochemistry (IHC),western blot, or fluorescence-activated cell sorting (FACS).

Histology of the liver showed uniform mCherry expression throughout thesection, while untreated animals did not express mCherry. Western blotswere also used to confirm mCherry expression in the treated animals,whereas mCherry was not detected in the untreated animals. Tubulin wasused as a control marker and was detected in both treated and untreatedmice, indicating that normal protein expression in hepatocytes wasunaffected.

FACS and IHC were also performed on the spleens of mCherry and untreatedmice. All leukocyte cell populations were negative for mCherryexpression by FACS analysis. By IHC, there were also no observabledifferences in the spleen in the spleen between mCherry treated anduntreated mice.

Example 31 Syringe Pump In Vivo Studies

mCherry modified mRNA (mRNA sequence shown in SEQ ID NO: 21439; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Capl) is formulated as a lipid nanoparticle (LNP) using the syringe pumpmethod. The LNP is formulated at a 20:1 weight ratio of total lipid tomodified mRNA with a final lipid molar ratio of 50:10:38.5:1.5(DLin-KC2-DMA: DSPC: Cholesterol: PEG-c-DOMG). The mCherry formulationis characterized by particle size, zeta potential, and encapsulation.

The LNP formulation is administered to mice (n=5) intravenously at amodified mRNA dose of 10 or 100 ug. Mice are sacrificed at 24 hrs afterdosing. The liver and spleen from the mice administered with mCherrymodified mRNA formulations are analyzed by immunohistochemistry (1HC),western blot, and/or fluorescence-activated cell sorting (FACS).

Example 32 In Vitro and In Vivo Expression

A. In vitro Expression in Human Cells Using Lipidoid Formulations

The ratio of mmRNA to lipidoid used to test for in vitro transfection istested empirically at different lipidoid:mmRNA ratios. Previous workusing siRNA and lipidoids have utilized 2.5:1, 5:1, 10:1, and 15:1lipidoid:siRNA wt:wt ratios. Given the longer length of mmRNA relativeto siRNA, a lower wt:wt ratio of lipidoid to mmRNA may be effective. Inaddition, for comparison mmRNA were also formulated using RNAIMAX™(Invitrogen, Carlsbad, Calif.) or TRANSIT-mRNA (Mirus Bio, Madison,Wis.) cationic lipid delivery vehicles.

The ability of lipidoid-formulated Luciferase (IVT cDNA sequence asshown in SEQ ID NO: 21445; mRNA sequence shown in SEQ ID NO: 21446,polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Capl, fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site), green fluorescentprotein (GFP) (IVT cDNA sequence as shown in SEQ ID NO: 21447; mRNAsequence shown in SEQ ID NO: 21448, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Capl, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site), G-CSF mRNA (mRNA sequence shown in SEQ ID NO: 21439;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Capl), and EPO mRNA (mRNA sequence shown in SEQ ID NO: 1638; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) to express the desired protein product can be confirmed byluminescence for luciferase expression, flow cytometry for GFPexpression, and by ELISA for G-CSF and Erythropoietin (EPO) secretion.

B. In vivo Expression Following Intravenous Injection

Systemic intravenous administration of the formulations are createdusing various different lipidoids including, but not limited to,98N12-5, C12-200, and MD1.

Lipidoid formulations containing mmRNA are injected intravenously intoanimals. The expression of the modified mRNA (mmRNA)-encoded proteinsare assessed in blood and/or other organs samples such as, but notlimited to, the liver and spleen collected from the animal. Conductingsingle dose intravenous studies will also allow an assessment of themagnitude, dose responsiveness, and longevity of expression of thedesired product.

In one embodiment, lipidoid based formulations of 98N12-5, C12-200, MD1and other lipidoids, are used to deliver luciferase, green fluorescentprotein (GFP), mCherry fluorescent protein, secreted alkalinephosphatase (sAP), human G-CSF, human Factor IX, or human Erythropoietin(EPO) mmRNA into the animal. After formulating mmRNA with a lipid, asdescribed previously, animals are divided into groups to receive eithera saline formulation, or a lipidoid-formulation which contains one of adifferent mmRNA selected from luciferase, GFP, mCherry, sAP, humanG-CSF, human Factor IX, and human EPO. Prior to injection into theanimal, mmRNA-containing lipidoid formulations are diluted in PBS.Animals are then administered a single dose of formulated mmRNA rangingfrom a dose of 10 mg/kg to doses as low as 1 ng/kg, with a preferredrange to be 10 mg/kg to 100 ng/kg, where the dose of mmRNA depends onthe animal body weight such as a 20 gram mouse receiving a maximumformulation of 0.2 ml (dosing is based no mmRNA per kg body weight).After the administration of the mmRNA-lipidoid formulation, serum,tissues, and/or tissue lysates are obtained and the level of themmRNA-encoded product is determined at a single and/or a range of timeintervals. The ability of lipidoid-formulated Luciferase, GFP, mCherry,sAP, G-CSF, Factor IX, and EPO mmRNA to express the desired proteinproduct is confirmed by luminescence for the expression of Luciferase,flow cytometry for the expression of GFP and mCherry expression, byenzymatic activity for sAP, or by ELISA for the section of G-CSF, FactorIX and/or EPO.

Further studies for a multi-dose regimen are also performed to determinethe maximal expression of mmRNA, to evaluate the saturability of themmRNA-driven expression (by giving a control and active mmRNAformulation in parallel or in sequence), and to determine thefeasibility of repeat drug administration (by giving mmRNA in dosesseparated by weeks or months and then determining whether expressionlevel is affected by factors such as immunogenicity). An assessment ofthe physiological function of proteins such as G-CSF and EPO are alsodetermined through analyzing samples from the animal tested anddetecting increases in granulocyte and red blood cell counts,respectively. Activity of an expressed protein product such as FactorIX, in animals can also be assessed through analysis of Factor IXenzymatic activity (such as an activated partial thromboplastin timeassay) and effect of clotting times.

C. In vitro Expression Following Intramuscular and/or SubcutaneousInjection

The use of lipidoid formulations to deliver oligonucleotides, includingmRNA, via an intramuscular route or a subcutaneous route of injectionneeds to be evaluated as it has not been previously reported.Intramuscular and/or subcutaneous injection of mmRNA are evaluated todetermine if mmRNA-containing lipidoid formulations are capabable toproduce both localized and systemic expression of a desired portiens.

Lipidoid formulations of 98N12-5, C12-200, and MD1 containing mmRNAselected from luciferase, green fluorescent protein (GFP), mCherryfluorescent protein, secreted alkaline phosphatase (sAP), human G-CSF,human factor IX, or human Erythropoietin (EPO) mmRNA are injectedintramuscularly and/or subcutaneously into animals. The expression ofmmRNA-encoded proteins are assessed both within the muscle orsubcutaneous tissue and systemically in blood and other organs such asthe liver and spleen. Single dose studies allow an assessment of themagnitude, dose responsiveness, and longevity of expression of thedesired product.

Animals are divided into groups to receive either a saline formulationor a formulation containing modified mRNA. Prior to injectionmmRNA-containing lipidoid formulations are diluted in PBS. Animals areadministered a single intramuscular dose of formulated mmRNA rangingfrom 50 mg/kg to doses as low as 1 ng/kg with a preferred range to be 10mg/kg to 100 ng/kg. A maximum dose for intramuscular administration, fora mouse, is roughly 1 mg mmRNA or as low as 0.02 ng mmRNA for anintramuscular injection into the hind limb of the mouse. Forsubcutaneous administration, the animals are administered a singlesubcutaneous dose of formulated mmRNA ranging from 400 mg/kg to doses aslow as 1 ng/kg with a preferred range to be 80 mg/kg to 100 ng/kg. Amaximum dose for subcutaneous administration, for a mouse, is roughly 8mg mmRNA or as low as 0.02 ng mmRNA.

For a 20 gram mouse the volume of a single intramuscular injection ismaximally 0.025 ml and a single subcutaneous injection is maximally 0.2ml. The optimal dose of mmRNA administered is calculated from the bodyweight of the animal. At various points in time points following theadministration of the mmRNA-lipidoid, serum, tissues, and tissue lysatesis obtained and the level of the mmRNA-encoded product is determined.The ability of lipidoid-formulated luciferase, green fluorescent protein(GFP), mCherry fluorescent protein, secreted alkaline phosphatase (sAP),human G-CSF, human factor IX, or human Erythropoietin (EPO) mmRNA toexpress the desired protein product is confirmed by luminescence forluciferase expression, flow cytometry for GFP and mCherry expression, byenzymatic activity for sAP, and by ELISA for G-CSF, Factor IX andErythropoietin (EPO) secretion.

Additional studies for a multi-dose regimen are also performed todetermine the maximal expression using mmRNA, to evaluate thesaturability of the mmRNA-driven expression (achieved by giving acontrol and active mmRNA formulation in parallel or in sequence), and todetermine the feasibility of repeat drug administration (by giving mmRNAin doses separated by weeks or months and then determining whetherexpression level is affected by factors such as immunogenicity). Studiesutilizing multiple subcutaneous or intramuscular injection sites at onetime point, are also utilized to further increase mmRNA drug exposureand improve protein production. An assessment of the physiologicalfunction of proteins, such as GFP, mCherry, sAP, human G-CSF, humanfactor IX, and human EPO, are determined through analyzing samples fromthe tested animals and detecting a change in granulocyte and/or redblood cell counts. Activity of an expressed protein product such asFactor IX, in animals can also be assessed through analysis of Factor IXenzymatic activity (such as an activated partial thromboplastin timeassay) and effect of clotting times.

Example 33 Bifunctional mmRNA

Using the teachings and synthesis methods described herein, modifiedRNAs are designed and synthesized to be bifunctional, thereby encodingone or more cytotoxic protein molecules as well as be synthesized usingcytotoxic nucleosides.

Administration of the bifunctional modified mRNAs is effected usingeither saline or a lipid carrier. Once administered, the bifunctionalmodified mRNA is translated to produce the encoded cytotoxic peptide.Upon degradation of the delivered modified mRNA, the cytotoxicnucleosides are released which also effect therapeutic benefit to thesubject.

Example 34 Modified mRNA Transfection

A. Reverse Transfection

For experiments performed in a 24-well collagen-coated tissue cultureplate, Keratinocytes are seeded at a cell density of 1×10⁵. Forexperiments performed in a 96-well collagen-coated tissue culture plate,Keratinocytes are seeded at a cell density of 0.5×10⁵. For each modifiedmRNA (mmRNA) to be transfected, modified mRNA: RNAIMAX™ is prepared asdescribed and mixed with the cells in the multi-well plate within aperiod of time, e.g., 6 hours, of cell seeding before cells had adheredto the tissue culture plate.

B. Forward Transfection

In a 24-well collagen-coated tissue culture plate, Keratinocytes areseeded at a cell density of 0.7×10⁵. For experiments performed in a96-well collagen-coated tissue culture plate, Keratinocytes are seededat a cell density of 0.3×10⁵. Keratinocytes are grown to a confluencyof >70% for over 24 hours. For each modified mRNA (mmRNA) to betransfected, modified mRNA: RNAIMAX™ is prepared as described andtransfected onto the cells in the multi-well plate over 24 hours aftercell seeding and adherence to the tissue culture plate.

C. Modified mRNA Translation Screen: G-CSF ELISA

Keratinocytes are grown in EPILIFE medium with Supplement S7 fromInvitrogen (Carlsbad, Calif.) at a confluence of >70%. One set ofkeratinocytes were reverse transfected with 300 ng of the chemicallymodified mRNA (mmRNA) complexed with RNAIMAX™ from Invitrogen. Anotherset of keratinocytes are forward transfected with 300 ng modified mRNAcomplexed with RNAIMAX™ from Invitrogen. The modified mRNA: RNAIMAX™complex is formed by first incubating the RNA with Supplement-freeEPILIFE® media in a 5× volumetric dilution for 10 minutes at roomtemperature.

In a second vial, RNAIMAX™ reagent was incubated with Supplement-freeEPILIFE® Media in 10× volumetric dilution for 10 minutes at roomtemperature. The RNA vial was then mixed with the RNAIMAX™ vial andincubated for 20-30 minutes at room temperature before being added tothe cells in a drop-wise fashion. Secreted human Granulocyte-ColonyStimulating Factor (G-CSF) concentration in the culture medium ismeasured at 18 hours post-transfection for each of the chemicallymodified mRNA in triplicate.

Secretion of Human G-CSF from transfected human keratinocytes isquantified using an ELISA kit from Invitrogen or R&D Systems(Minneapolis, Minn.) following the manufacturers recommendedinstructions.

D. Modified mRNA Dose and Duration: G-CSF ELISA

Keratinocytes are grown in EPILIFE® medium with Supplement S7 fromInvitrogen at a confluence of >70%. Keratinocytes are reversetransfected with either 0 ng, 46.875 ng, 93.75 ng, 187.5 ng, 375 ng, 750ng, or 1500 ng modified mRNA complexed with the RNAIMAX™ from Invitrogen(Carlsbad, Calif.). The modified mRNA:RNAIMAX™ complex is formed asdescribed. Secreted human G-CSF concentration in the culture medium ismeasured at 0, 6, 12, 24, and 48 hours post-transfection for eachconcentration of each modified mRNA in triplicate. Secretion of humanG-CSF from transfected human keratinocytes is quantified using an ELISAkit from Invitrogen or R&D Systems following the manufacturersrecommended instructions.

Example 35 Split Dose Studies

Studies utilizing multiple subcutaneous or intramuscular injection sitesat one time point were designed and performed to investigate ways toincrease mmRNA drug exposure and improve protein production. In additionto detection of the expressed protein product, an assessment of thephysiological function of proteins was also determined through analyzingsamples from the animal tested.

Surprisingly, it has been determined that split dosing of mmRNA producesgreater protein production and phenotypic responses than those producedby single unit dosing or multi-dosing schemes.

The design of a single unit dose, multi-dose and split dose experimentinvolved using human erythropoietin (EPO) mmRNA (mRNA shown in SEQ IDNO: 1638; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Capl) administered in buffer alone. The dosing vehicle(F. buffer) consisted of 150 mM NaCl, 2 mM CaCl₂, 2 mM Na⁺-phosphate(1.4 mM monobasic sodium phosphate; 0.6 mM dibasic sodium phosphate),and 0.5 mM EDTA, pH 6.5. The pH was adjusted using sodium hydroxide andthe final solution was filter sterilized. The mmRNA was modified with5meC at each cytosine and pseudouridine replacement at each uridinesite.

Animals (n=5) were injected IM (intramuscular) for the single unit doseof 100 ug. For multi-dosing, two schedules were used, 3 doses of 100 ugand 6 doses of 100 ug. For the split dosing scheme, two schedules wereused, 3 doses at 33.3 ug and 6 doses of 16.5 ug mmRNA. Control dosinginvolved use of buffer only at 6 doses. Control mmRNA involved the useof luciferase mmRNA (IVT cDNA sequence shown in SEQ ID NO: 21445; mRNAsequence shown in SEQ ID NO: 21446, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Capl, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) dosed 6 times at 100 ug. Blood and muscle tissue wereevaluated 13 hrs post injection.

Human EPO protein was measured in mouse serum 13h post I.M. single,multi- or split dosing of the EPO mmRNA in buffer. Seven groups of mice(n=5 mice per group) were treated and evaluated. The results are shownin Table 60.

TABLE 60 Split dose study Avg. Polypeptide pmol/mL per unit Dose Dose ofTotal human drug Splitting Group Treatment mmRNA Dose EPO (pmol/ug)Factor 1 Human EPO 1 × 100 ug 100 ug 14.3 .14 1 mmRNA 2 Human EPO 3 ×100 ug 300 ug 82.5 .28 2 mmRNA 3 Human EPO 6 × 100 ug 600 ug 273.0 .463.3 mmRNA 4 Human EPO 3 × 33.3 ug  100 ug 104.7 1.1 7.9 mmRNA 5 HumanEPO 6 × 16.5 ug  100 ug 127.9 1.3 9.3 mmRNA 6 Luciferase mmRNA 6 × 100ug 600 ug 0 — — 7 Buffer Alone — — 0 — —

The splitting factor is defined as the product per unit drug divided bythe single dose product per unit drug (PUD). For example for treatmentgroup 2 the value 0.28 or product (EPO) per unit drug (mmRNA) is dividedby the single dose product per unit drug of 0.14. The result is 2.Likewise, for treatment group 4, the value 1.1 or product (EPO) per unitdrug (mmRNA) is divided by the single dose product per unit drug of0.14. The result is 7.9. Consequently, the dose splitting factor (DSF)may be used as an indicator of the efficacy of a split dose regimen. Forany single administration of a total daily dose, the DSF should be equalto 1. Therefore any DSF greater than this value in a split dose regimenis an indication of increased efficacy.

To determine the dose response trends, impact of injection site andimpact of injection timing, studies are performed. In these studies,varied doses of 1 ug, 5 ug, 10 ug, 25 ug, 50 ug, and values in betweenare used to determine dose response outcomes. Split dosing for a 100 ugtotal dose includes three or six doses of 1.6 ug, 4.2 ug, 8.3 ug, 16.6ug, or values and total doses equal to administration of the total doseselected.

Injection sites are chosen from the limbs or any body surface presentingenough area suitable for injection. This may also include a selection ofinjection depth to target the dermis (Intradermal), epidermis(Epidermal), subcutaneous tissue (SC) or muscle (IM). Injection anglewill vary based on targeted delivery site with injections targeting theintradermal site to be 10-15 degree angles from the plane of the surfaceof the skin, between 20-45 degrees from the plane of the surface of theskin for subcutaneous injections and angles of between 60-90 degrees forinjections substantially into the muscle.

Example 36 Quantification in Exosomes

The quantity and localization of the mmRNA of the present invention canbe determined by measuring the amounts (initial, timecourse, or residualbasis) in isolated exosomes. In this study, since the mmRNA aretypically codon-optimized and distinct in sequence from endogenous mRNA,the levels of mmRNA are quantitated as compared to endogenous levels ofnative or wild type mRNA by using the methods of Gibbings,PCT/IB2009/005878, the contents of which are incorporated herein byreference in their entirety.

In these studies, the method is performed by first isolating exosomes orvesicles preferably from a bodily fluid of a patient previously treatedwith a polynucleotide, primary construct or mmRNA of the invention, thenmeasuring, in said exosomes, the polynucleotide, primary construct ormmRNA levels by one of mRNA microarray, qRT-PCR, or other means formeasuring RNA in the art including by suitable antibody orimmunohistochemical methods.

Example 37 Effect of Modified mRNA on Cellular Viability, Cytotoxicityand Apoptosis

This experiment demonstrates cellular viability, cytotoxicity andapoptosis for distinct modified mRNA in-vitro transfected HumanKeratinocyte cells. Keratinocytes are grown in EPILIFE® medium withHuman Keratinocyte Growth Supplement in the absence of hydrocortisonefrom Invitrogen (Carlsbad, Calif.) at a confluence of >70%.Keratinocytes are reverse transfected with 0 ng, 46.875 ng, 93.75 ng,187.5 ng, 375 ng, 750 ng, 1500 ng, 3000 ng, or 6000 ng of modified mRNAcomplexed with RNAIMAX™ from Invitrogen. The modified mRNA:RNAIMAX™complex is formed. Secreted human G-CSF concentration in the culturemedium is measured at 0, 6, 12, 24, and 48 hours post-transfection foreach concentration of each modified m RNA in triplicate. Secretion ofhuman G-CSF from transfected human keratinocytes is quantified using anELISA kit from Invitrogen or R&D Systems following the manufacturersrecommended instructions.

Cellular viability, cytotoxicity and apoptosis is measured at 0, 12, 48,96, and 192 hours post-transfection using the APOTOX-GLO™ kit fromPromega (Madison, Wis.) according to manufacturer instructions.

Example 38 Detection of a Cellular Innate Immune Response to ModifiedmRNA Using an ELISA Assay

An enzyme-linked immunosorbent assay (ELISA) for Human Tumor NecrosisFactor-α (TNF-α), Human Interferon-β (IFN-β) and HumanGranulocyte-Colony Stimulating Factor (G-CSF) secreted from invitro-transfected Human Keratinocyte cells is tested for the detectionof a cellular innate immune response. Keratinocytes are grown inEPILIFE® medium with Human Keratinocyte Growth Supplement in the absenceof hydrocortisone from Invitrogen (Carlsbad, Calif.) at a confluenceof >70%. Secreted TNF-α keratinocytes are reverse transfected with 0 ng,93.75 ng, 1 87.5 ng, 375 ng, 750 ng, 1500 ng or 3000 ng of thechemically modified mRNA (mmRNA) complexed with RNAIMAX™ from Invitrogenas described in triplicate. Secreted TNF-α in the culture medium ismeasured 24 hours post-transfection for each of the chemically modifiedmRNA using an ELISA kit from Invitrogen according to the manufacturerprotocols.

Secreted IFN-β in the same culture medium is measured 24 hourspost-transfection for each of the chemically modified mRNA using anELISA kit from Invitrogen according to the manufacturer protocols.Secreted human G-CSF concentration in the same culture medium ismeasured at 24 hours post-transfection for each of the chemicallymodified mRNA. Secretion of human G-CSF from transfected humankeratinocytes is quantified using an ELISA kit from Invitrogen or R&DSystems (Minneapolis, Minn.) following the manufacturers recommendedinstructions. These data indicate which modified mRNA (mmRNA) arecapable eliciting a reduced cellular innate immune response incomparison to natural and other chemically modified polynucleotides orreference compounds by measuring exemplary type 1 cytokines TNF-α andIFN-β.

Example 39 Human Granulocyte—Colony Stimulating Factor (G-CSF) ModifiedmRNA-induced Cell Proliferation Assay

Human keratinocytes are grown in EPILIFE® medium with Supplement S7 fromInvitrogen at a confluence of >70% in a 24-well collagen-coatedTRANSWELL® (Coming, Lowell, Mass.) co-culture tissue culture plate.Keratinocytes are reverse transfected with 750 ng of the indicatedchemically modified mRNA (mmRNA) complexed with RNAIMAX from Invitrogenas described in triplicate. The modified mRNA:RNAIMAX complex is formedas described. Keratinocyte media is exchanged 6-8 hourspost-transfection. 42-hours post-transfection, the 24-well TRANSWELL®plate insert with a 0.4 tm-pore semi-permeable polyester membrane isplaced into the human G-CSF modified mRNA-transfected keratinocytecontaining culture plate

Human myeloblast cells, Kasumi-1 cells or KG-1 (0.2×10⁵ cells), areseeded into the insert well and cell proliferation is quantified 42hours post-co-culture initiation using the CyQuant Direct CellProliferation Assay (Invitrogen, Carlsbad, Calif.) in a 100-120 μlvolume in a 96-well plate. Modified mRNA-encoding human G-CSF-inducedmyeloblast cell proliferation is expressed as a percent cellproliferation normalized to untransfected keratinocyte/myeloblastco-culture control wells. Secreted human G-CSF concentration in both thekeratinocyte and myeloblast insert co-culture wells is measured at 42hours post-co-culture initiation for each modified mRNA in duplicate.Secretion of human G-CSF is quantified using an ELISA kit fromInvitrogen following the manufacturer recommended instructions.

Transfected human G-CSF modified mRNA in human keratinocyte feeder cellsand untransfected human myeloblast cells are detected by RT-PCR. TotalRNA from sample cells is extracted and lysed using RNEASY® kit (Qiagen,Valencia, Calif.) according to the manufacturer instructions. Extractedtotal RNA is submitted to RT-PCR for specific amplification of modifiedmRNA-G-CSF using PROTOSCRIPT® M-MuLV Taq RT-PCR kit (New EnglandBioLabs, Ipswich, Mass.) according to the manufacturer instructions withhuman G-CSF-specific primers. RT-PCR products are visualized by 1.2%agarose gel electrophoresis.

Example 40 Co-Culture Assay

Modified mRNA comprised of chemically-distinct modified nucleotidesencoding human Granulocyte-Colony Stimulating Factor (G-CSF) maystimulate the cellular proliferation of a transfection incompetent cellin a co-culture environment. The co-culture includes a highlytransfectable cell type such as a human keratinocyte and a transfectionincompetent cell type such as a white blood cell (WBC). The modifiedmRNA encoding G-CSF are transfected into the highly transfectable cellallowing for the production and secretion of G-CSF protein into theextracellular environment where G-CSF acts in a paracrine-like manner tostimulate the white blood cell expressing the G-CSF receptor toproliferate. The expanded WBC population may be used to treatimmune-compromised patients or partially reconstitute the WBC populationof an immunosuppressed patient and thus reduce the risk of opportunisticinfections.

In another example, a highly transfectable cell such as a fibroblast aretransfected with certain growth factors support and simulate the growth,maintenance, or differentiation of poorly transfectable embryonic stemcells or induced pluripotent stem cells.

Example 41 Detection Assays of Human IgG Antibodies

A. ELISA Detection of Human IgG Antibodies

This example describes an ELISA for Human IgG from Chinese HamsterOvary's (CHO) and Human Embryonic Kidney (HEK, HER-2 Negative) 293 cellstransfected with human IgG modified mRNA (mmRNA). The Human EmbryonicEmbryonic Kidney (HEK) 293 are grown in CD 293 Medium with Supplement ofL-Glutamine from Invitrogen until they reach a confluence of 80-90%. TheCHO cells are grown in CD CHO Medium with Supplement of L-Glutamine,Hypoxanthine and Thymidine. In one aspect, 2×106 cells are transfectedwith 24 μg modified mRNA complexed with RNAIMAX™ from Invitrogen in a 75cm2 culture flask from Corning in 7 ml of medium. In another aspect,80,000 cells are transfected with 1 μg modified mRNA complexed withRNAIMAX™ from Invitrogen in a 24-well plate. The modified mRNA:RNAIMAX™complex is formed by incubating in a vial the mmRNA with either the CD293 or CD CHO medium in a 5× volumetric dilution for 10 minutes at roomtemperature. In a second vial, RNAIMAX™ reagent is incubated with CD 293medium or CD CHO medium in a 10× volumetric dilution for 10 minutes atroom temperature. The mmRNA vial is then mixed with the RNAIMAX™ vialand incubated for 20-30 minutes at room temperature before it is addedto the CHO or HEK cells in a drop-wise fashion. The culture supernatantsare stored at 4 degrees celsius. The concentration of the secreted humanIgG in the culture medium in the 24 μg mmRNA transfections is measuredat 12, 24, 36 hours post-transfection and the 1 μg mmRNA transfection ismeasured at 36 hours. Secretion of Trastuzumab from transfected HEK 293cells is quantified using an ELISA kit from Abcam (Cambridge, Mass.)following the manufacturers recommended instructions. The data showsthat a Humanized IgG antibody (such as Trastuzumab) mmRNA is capable ofbeing translated in HEK Cells and that Trastuzumab is secreted out ofthe cells and released into the extracellular environment. Furthermore,the data demonstrate that transfection of cells with mmRNA encodingTrastuzumab for the production of secreted protein can be scaled up to abioreactor or large cell culture conditions.

B. Western Detection of Modified mRNA Produced Human IgG Antibody

A Western Blot of CHO-K1 cells is co-transfected with 1 μg each of Heavyand Light Chain of Trastuzumab modified mRNA (mmRNA). CHO cells aregrown using standard protocols in 24-well plates. The cell supernatantsor cell lysates are collected 24 hours post-transfection, separated on a12% SDS-Page gel and transferred onto a nitrocellulose membrane usingthe IBOT® by Invitrogen (Carlsbad, Calif.). The cells are incubated witha first conjugation of a rabbit polyclonal antibody to Human IgGconjugated to DYLIGHT594 (ab96904, abcam, Cambridge, Mass.) and a secondconjugation of a goat polyclonal antibody to Rb IgG which is conjugatedto alkaline phosphatase. After incubation, the antibody is detectedusing Novex® alkaline phosphatase chromogenic substrate by Invitrogen(Carlsbad, Calif.).

C. Cell Immuno Staining of Modified mRNA Produced Trastuzumab andRituximab

CHO-K1 cells are co-transfected with 500 ng each of Heavy and LightChain of either Trastuzumab or Rituximab. Cells are grown in F-12KMedium from GIBCO® (Grand Island, N.Y.) and 10% FBS. Cells are fixedwith 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 inPBS for 5-10 minutes at room temperature and cells are washed 3 timeswith room temperature PBS. Trastuzumab and Rituximab staining isperformed using rabbit polyclonal antibody to Human IgG conjugated toDYLIGHT®594 (ab96904, abcam, Cambridge, Mass.) according to themanufacture's recommended dilutions. Nuclear DNA staining is performedwith DAPI dye from Invitrogen (Carlsbad, Calif.). The protein forTrastuzumab and Rituximab is translated and localized to the cytoplasmupon modified mRNA transfections. Pictures are taken 13 hourspost-transfection.

D. Binding Immunoblot Assay for Modified mRNA Produced Trastuzumab andRituximab

Trastuzumab and Rituximab are detected using a binding immunoblotdetection assay. Varying concentrations (100 ng/ul to 0 ng/ul) of theErB2 peptide (ab40048, abeam, Cambridge, Mass.), antigen for Trastuzumaband the CD20 peptide (ab97360, abeam, Cambridge, Mass.), antigen forRituximab are run on a 12% SDS-Page gel and transferred onto a membraneusing the iBlot from Invitrogen. The membranes are incubated for 1 hourwith their respective cell supernatants from CHO-K1 cells which areco-transfected with 500 ng each of Heavy and Light Chain of eitherTrastuzumab or Rituximab. The membranes are blocked with 1% BSA and asecondary anti-human IgG antibody conjugated to alkaline phosphatase(abcam, Cambridge, Mass.) is added. Antibody detection is conductedusing the NOVEX alkaline phosphatase chromogenic substrate by Invitrogen(Carlsbad, Calif.). The data shows that a humanized IgG antibodiesgenerated from modified mRNA is capable of recognizing and binding totheir respective antigens.

E. Cell Proliferation Assay

The SK-BR-3 cell line, an adherent cell line derived from a human breastadenocarcinoma, which overexpresses the HER2/neu receptor can be used tocompare the anti-proliferative properties of modified mRNA (mmRNA)generated Trastuzumab. Varying concentrations of purified Trastuzumabgenerated from modified mRNA and trastuzumab are be added to cellcultures, and their effects on cell growth are be assessed in triplicatecytotoxicity and viability assays.

Example 42 Bulk Transfection of Modified mRNA into Cell Culture

A. Cationic Lipid Delivery Vehicles

RNA transfections are carried out using RNAIMAX™ (Invitrogen, Carlsbad,Calif.) or TRANSIT-mRNA (Mirus Bio, Madison, Wis.) cationic lipiddelivery vehicles. RNA and reagent are first diluted in Opti-MEM basalmedia (Invitrogen, Carlsbad, Calif.). 100 ng/uL RNA is diluted 5× and 5μL, of RNAIMax per μg of RNA is diluted 10×. The diluted components arepooled and incubated 15 minutes at room temperature before they aredispensed to culture media. For TRANSIT-mRNA transfections, 100 ng/uLRNA is diluted 10× in Opti-MEM and BOOST reagent is added (at aconcentration of 2 μL, per μg of RNA), TRANSIT-mRNA is added (at aconcentration of 2 μL, per μg of RNA), and then the RNA-lipid complexesare delivered to the culture media after a 2-minute incubation at roomtemperature. RNA transfections are performed in Nutristem xenofree hESmedia (Stemgent, Cambridge, Mass.) for RiPS derivations, Dermal CellBasal Medium plus Keratinocyte Growth Kit (ATCC) for keratinocyteexperiments, and Opti-MEM plus 2% FBS for all other experiments.Successful introduction of a modified mRNA (mmRNA) into host cells canbe monitored using various known methods, such as a fluorescent marker,such as Green Fluorescent Protein (GFP). Successful transfection of amodified mRNA can also be determined by measuring the protein expressionlevel of the target polypeptide by e.g., Western Blotting orimmunocytochemistry. Similar methods may be followed for large volumescale-up to multi-liter (5-10,000L) culture format following similarRNA-lipid complex ratios.

B. Electroporation Delivery of Exogenous Synthetic mRNA Transcripts

Electroporation parameters are optimized by transfecting MRC-5fibroblasts with in vitro synthetic modified mRNA (mmRNA) transcriptsand measuring transfection efficiency by quantitative RT-PCR withprimers designed to specifically detect the exogenous transcripts.Discharging a 150 uF capacitor charged to F into 2.5×106 cells suspendedin 50 μl of Opti-MEM (Invitrogen, Carlsbad, Calif.) in a standardelectroporation cuvette with a 2 mm gap is sufficient for repeateddelivery in excess of 10,000 copies of modified mRNA transcripts percell, as determined using the standard curve method, while maintaininghigh viability (>70%). Further experiments may reveal that the voltagerequired to efficiently transfect cells with mmRNA transcripts candepend on the cell density during electroporation. Cell density may varyfrom 1×10⁶ cell/50 μl to a density of 2.5×10⁶ cells/50 μl and requirefrom 110V to 145V to transfect cells with similar efficiencies measuredin transcript copies per cell. Large multi-liter (5-10,000 L)electroporation may be performed similar to large volume flowelectroporation strategies similar to methods described with the abovedescribed constraints (Li et al., 2002; Geng et al., 2010).

Example 43 In Vivo Delivery Using Lipoplexes

A. Human EPO Modified RNA Lipoplex

A formulation containing 100 μg of modified human erythropoietinmRNA(mRNA shown in SEQ ID NO: 1638; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) (EPO; fully modified5-methylcytosine; N1-methyl-pseudouridine) was lipoplexed with 30% byvolume of RNAIMAX™ (Lipoplex-h-Epo-46; Generation 2 or Gen2) in 50-70 uLdelivered intramuscularly to four C57/BL6 mice. Other groups consistedof mice receiving an injection of the lipoplexed modified luciferasemRNA (Lipoplex-luc) (IVT cDNA sequence shown in SEQ ID NO: 21445; mRNAsequence shown in SEQ ID NO: 21446, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Capl, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) which served as a control containing 100 μg of modifiedluciferase mRNA was lipoplexed with 30% by volume of RNAiMAX™ or micereceiving an injection of the formulation buffer as negative control ata dose volume of 65 ul. 13 hours after the intramuscular injection,serum was collected from each mouse to measure the amount of human EPOprotein in the mouse serum by human EPO ELISA and the results are shownin Table 61.

TABLE 61 Human EPO Production (IM Injection Route) Formualtion AverageLipoplex-h-Epo-46 251.95 Lipoplex-Luc 0 Formulation Buffer 0

B. Human G-CSF Modified RNA Lipoplex

A formulation containing 100 μg of one of two versions of modified humanG-CSF mRNA(mRNA sequence shown in SEQ ID NO: 21438; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1)(G-CSF fully modified with 5-methylcytosine and pseudouridine (G-CSF) orG-CSF fully modified with 5-methylcytosine and N1-methyl-pseudouridine(G-CSF-N1) lipoplexed with 30% by volume of RNAIMAX™ and delivered in150 uL intramuscularly (I.M), in 150 uL subcutaneously (S.C) and in 225uL intravenously (I.V) to C57/BL6 mice.

Three control groups were administered either 100 μg of modifiedluciferase mRNA (IVT cDNA sequence shown in SEQ ID NO: 21445; mRNAsequence shown in SEQ ID NO: 21446, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Capl, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) intramuscularly (Luc-unsp I.M.) or 150 μg of modifiedluciferase mRNA intravenously (Luc-unsp I.V.) or 150 uL of theformulation buffer intramuscularly (Buffer I.M.). 6 hours afteradministration of a formulation, serum was collected from each mouse tomeasure the amount of human G-CSF protein in the mouse serum by humanG-CSF ELISA and the results are shown in Table 62.

These results demonstrate that both 5-methylcytosine/pseudouridine and5-methylcytosine/N1-methyl-pseudouridine modified human G-CSF mRNA canresult in specific human G-CSF protein expression in serum whendelivered via I.V. or I.M. route of administration in a lipoplexformulation.

TABLE 62 Human G-CSF in Serum (I.M., I.V., S.C. Injection Route)Formulation Route G-CSF (pg/ml) G-CSF I.M. 85.6 G-CSF N1 I.M. 40.1 G-CSFS.C. 3.9 G-CSF N1 S.C. 0.0 G-CSF I.V. 31.0 G-CSF N1 I.V. 6.1 Luc-unspI.M. 0.0 Luc-unsp I.V. 0.0 Buffer I.M. 0.0

C. Human G-CSF Modified RNA Lipoplex Comparison

A formulation containing 100 μg of either modified human G-CSF mRNAlipoplexed with 30% by volume of RNAIMAX™ with a 5-methylcytosine (5mc)and a pseudouridine (ψ) modification (G-CSF-Gen1-Lipoplex), modifiedhuman G-CSF mRNA with a 5mc and ψ modification in saline(G-CSF-Genl-Saline), modified human G-CSF mRNA with aN1-5-methylcytosine (N1-5mc) and a ψ modification lipoplexed with 30% byvolume of RNAIMAX™ (G-CSF-Gent-Lipoplex), modified human G-CSF mRNA witha N1-5mc and ψ modification in saline (G-CSF-Gent-Saline), modifiedluciferase with a 5mc and ψ modification lipoplexed with 30% by volumeof RNAIMAX™ (Luc-Lipoplex), or modified luciferase mRNA with a a 5mc andψ modification in saline (Luc-Saline) was delivered intramuscularly(I.M.) or subcutaneously (S.C.) and a control group for each method ofadministration was giving a dose of 80 uL of the formulation buffer (F.Buffer) to C57/BL6 mice. 13 hours post injection serum and tissue fromthe site of injection were collected from each mouse and analyzed byG-CSF ELISA to compare human G-CSF protein levels. The results of thehuman G-CSF protein in mouse serum from the intramuscularadministration, and the subcutaneous administration results are shown inTable 63.

These results demonstrate that 5-methylcytosine/pseudouridine and5-methylcytosine/N1-methyl-pseudouridine modified human G-CSF mRNA canresult in specific human G-CSF protein expression in serum whendelivered via I.M. or S.C. route of administration whether in a salineformulation or in a lipoplex formulation. As shown in Table 63,5-methylcytosine/N1-methyl-pseudouridine modified human G-CSF mRNAgenerally demonstrates increased human G-CSF protein production relativeto 5-methylcytosine/pseudouridine modified human G-CSF mRNA.

TABLE 63 Human G-CSF Protein in Mouse Serum G-CSF (pg/ml) FormulationI.M. Injection Route S.C. Injenction Route G-CSF-Gen1-Lipoplex 13.98842.855 G-CSF-Gen1-saline 9.375 4.614 G-CSF-Gen2-lipoplex 75.572 32.107G-CSF-Gen2-saline 20.190 45.024 Luc lipoplex 0 3.754 Luc saline 0.0748 0F. Buffer 4.977 2.156

D. mCherry Modified RNA Lipoplex Comparison

Intramuscular and Subcutaneous Administration

A formulation containing 100 μg of either modified mCherry mRNA (mRNAsequence shown in SEQ ID NO: 21439; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) lipoplexed with 30% byvolume of RNAIMAX™ or modified mCherry mRNA in saline is deliveredintramuscularly and subcutaneously to mice. A formulation buffer is alsoadministered to a control group of mice either intramuscularly orsubcutaneously. The site of injection on the mice may be collected 17hours post injection for sectioning to determine the cell type(s)responsible for producing protein.

Intravitreal Administration

A formulation containing 10 μg of either modified mCherry mRNAlipoplexed with RNAIMAX™, modified mCherry mRNA in a formulation buffer,modified luciferase mRNA lipoplexed with RNAMAX™, modified luciferasemRNA in a formulation buffer can be administered by intravitrealinjection (IVT) in rats in a dose volume of 5 μl/eye. A formulationbuffer is also administrered by IVT to a control group of rats in a dosevolume of 5 μl/eye. Eyes from treated rats can be collected after 18hours post injection for sectioning and lysating to determine whethermmRNA can be effectively delivered in vivo to the eye and result inprotein production, and to also determine the cell type(s) responsiblefor producing protein in vivo.

Intranasal Administration

A formulation containing 100 μg of either modified mCherry mRNAlipoplexed with 30% by volume of RNAIMAX™, modified mCherry mRNA insaline, modified luciferase mRNA lipoplexed with 30% by volume ofRNAIMAX™ or modified luciferase mRNA in saline is deliveredintranasally. A formulation buffer is also administered to a controlgroup intranasally. Lungs may be collected about 13 hours postinstillation for sectioning (for those receiving mCherry mRNA) orhomogenization (for those receiving luciferase mRNA). These samples willbe used to determine whether mmRNA can be effectively delivered in vivoto the lungs and result in protein production, and to also determine thecell type(s) responsible for producing protein in vivo.

Example 44 In Vivo Delivery Using Varying Lipid Ratios

Modified mRNA was delivered to C57/BL6 mice to evaluate varying lipidratios and the resulting protein expression. Formulations of 100 μgmodified human EPO mRNA (mRNA shown in SEQ ID NO: 1638; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) lipoplexed with 10%,30% or 50% RNAIMAX™, 100 μg modified luciferase mRNA (IVT cDNA sequenceshown in SEQ ID NO: 21445; mRNA sequence shown in SEQ ID NO: 21446,polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site) lipoplexed with 10%, 30%or 50% RNAIMAX™ or a formulation buffer were administeredintramuscularly to mice in a single 70 μl dose. Serum was collected 13hours post injection to undergo a human EPO ELISA to determine the humanEPO protein level in each mouse. The results of the human EPO ELISA,shown in Table 64, show that modified human EPO expressed in the muscleis secreted into the serum for each of the different percentage ofRNAIMAX™.

TABLE 64 Human EPO Protein in Mouse Serum (IM Injection Route)Formulation EPO (pg/ml) Epo + 10% RNAiMAX 11.4 Luc + 10% RNAiMAX 0 Epo +30% RNAiMAX 27.1 Luc + 30% RNAiMAX 0 Epo + 50% RNAiMAX 19.7 Luc + 50%RNAiMAX 0 F. Buffer 0

Example 45 Intramuscular and Subcutaneous In Vivo Delivery in Mammals

Modified human EPO mRNA (mRNA sequence shown in SEQ ID NO: 1638; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) formulatedin formulation buffer was delivered to either C57/BL6 mice orSprague-Dawley rats to evaluate the dose dependency on human EPOproduction. Rats were intramuscularlly injected with 50 μl of themodified human EPO mRNA (h-EPO), modified luciferase mRNA (Luc) (IVTcDNA sequence shown in SEQ ID NO: 21445; mRNA sequence shown in SEQ IDNO: 21446, polyA tail of approximately 160 nucleotides not shown insequence, 5′ cap, Cap1, fully modified with 5-methylcytosine at eachcytosine and pseudouridine replacement at each uridine site) or theformulation buffer (F.Buffer) as described in the dosing chart Table 65.

Mice were intramuscularly or subcutaneously injected with 50 μl of themodified human EPO mRNA (h-EPO), modified luciferase mRNA (Luc) or theformulation buffer (F.Buffer) as described in the dosing chart Table 66.13 hours post injection blood was collected and serum was analyzed todetermine the amount human EPO for each mouse or rat. The average andgeometric mean in pg/ml for the rat study are also shown in Table 65.

TABLE 65 Rat Study Dose Avg. Geometric- Group (ug) pg/ml mean pg/mlh-EPO G#1 150 67.7 67.1 h-EPO G#2 100 79.4 66.9 h-EPO G#3 50 101.5 85.4h-EPO G#4 10 46.3 31.2 h-EPO G#5 1 28.7 25.4 Luc G#6 100 24.5 22.4 F.Buffer G#7 — 18.7 18.5

TABLE 66 Mouse Study Average Level Route Treatment Group Dose in serumpg/ml IM h-EPO 1 100 μg 96.2 IM h-EPO 2  50 μg 63.5 IM h-EPO 3  25 μg18.7 IM h-EPO 4  10 μg 25.9 IM h-EPO 5  1 μg 2.6 IM Luc 6 100 μg 0 IM F.Buffer 7 — 1.0 SC h-EPO 1 100 μg 72.0 SC Luc 2 100 μg 26.7 SC F. Buffer3 — 17.4

Example 46 Duration of Activity after Intramuscular In Vivo Delivery

Modified human EPO mRNA (mRNA sequence shown in SEQ ID NO: 1638; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) formulatedin formulation buffer was delivered to Sprague-Dawley rats to determinethe duration of the dose response. Rats were intramuscularly injectedwith 50 μl of the modified human EPO mRNA (h-EPO), modified luciferasemRNA(IVT cDNA sequence shown in SEQ ID NO: 21445; mRNA sequence shown inSEQ ID NO: 21446, polyA tail of approximately 160 nucleotides not shownin sequence, 5′ cap, Cap1, fully modified with 5-methylcytosine at eachcytosine and pseudouridine replacement at each uridine site) (Luc) orthe formulation buffer (F.Buffer) as described in the dosing chart Table67. The rats were bled 2, 6, 12, 24, 48 and 72 hours after theintramuscular injection to determine the concentration of human EPO inserum at a given time. The average and geometric mean in pg/ml for thisstudy are also shown in Table 67.

TABLE 67 Dosing Chart Dose Avg. Geometric- Group (ug) pg/ml mean (pg/ml)h-EPO  2 hour 100 59.6 58.2 h-EPO  6 hour 100 68.6 55.8 h-EPO 12 hour100 87.4 84.5 h-EPO 24 hour 100 108.6 95.3 h-EPO 48 hour 100 77.9 77.0h-EPO 72 hour 100 80.1 75.8 Luc 24, 48 100 37.2 29.2 and 72 hour F.Buffer 24, 48 — 48.9 10.4 and 72 hour

Example 47 Routes of Administration

Further studies were performed to investigate dosing using differentroutes of administration. Following the protocol outlined in Example 35,4 mice per group were dosed intramuscularly (I.M.), intravenously (IV)or subcutaneously (S.C.) by the dosing chart outlined in Table 68. Serumwas collected 13 hours post injection from all mice, tissue wascollected from the site of injection from the intramuscular andsubcutaneous group and the spleen, liver and kidneys were collected fromthe intravenous group. The results from the intramuscular group and thesubcutaneous group results are shown in Table 69.

TABLE 68 Dosing Chart Total Dosing Group Treatment Route Dose of mmRNADose Vehicle 1 Lipoplex-human EPO mmRNA I.M. 4 x 100 ug + 30% 4x70 ulLipoplex Lipoplex 2 Lipoplex-human EPO mmRNA I.M. 4 x 100 ug 4x70 ulBuffer 3 Lipoplex-human EPO mmRNA S.C. 4 x 100 ug + 30% 4x70 ul LipoplexLipoplex 4 Lipoplex-human EPO mmRNA S.C. 4 x 100 ug 4x70 ul Buffer 5Lipoplex-human EPO mmRNA I.V. 200 ug + 30% Lipoplex 140 ul Lipoplex 6Lipoplexed-Luciferase mmRNA I.M. 100 ug + 30% Lipoplex 4x70 ul Lipoplex7 Lipoplexed-Luciferase mmRNA I.M. 100 ug 4x70 ul Buffer 8Lipoplexed-Luciferase mmRNA S.C. 100 ug + 30% Lipoplex 4x70 ul Lipoplex9 Lipoplexed-Luciferase mmRNA S.C. 100 ug 4x70 ul Buffer 10Lipoplexed-human EPO mmRNA I.V. 200 ug + 30% Lipoplex 140 ul Lipoplex 11Formulation Buffer I.M. 4x multi dosing 4x70 ul Buffer

TABLE 69 Human EPO Protein in Mouse Serum (I.M. Injection Route) EPO(pg/ml) Formulation I.M. Injection Route S.C. Injection RouteEpo-Lipoplex 67.115 2.154 Luc-Lipoplex 0 0 Epo-Saline 100.891 11.37Luc-Saline 0 0 Formulation Buffer 0 0

Example 48 Rapidly Eliminated Lipid Nanoparticle (reLNP) Studies

A. Formulation of Modified RNA reLNPs

Solutions of synthesized lipid, 1,2-distearoyl-3-phosphatidylcholine(DSPC)

(Avanti Polar Lipids, Alabaster, Ala.), cholesterol (Sigma-Aldrich,Taufkirchen, Germany), andα-[3′-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-ω-methoxy-polyoxyethylene(PEG-c-DOMG) (NOF, Bouwelven, Belgium) are prepared and stored at −20°C. The synthesized lipid is selected from DLin-DMA with an internalester, DLin-DMA with a terminal ester, DLin-MC3-DMA-internal ester, andDLin-MC3-DMA with a terminal ester. The reLNPs are combined to yield amolar ratio of 50:10:38.5:1.5 (reLNP: DSPC: Cholesterol: PEG-c-DOMG).Formulations of the reLNPs and modified mRNA are prepared by combiningthe lipid solution with the modified mRNA solution at total lipid tomodified mRNA weight ratio of 10:1, 15:1, 20:1 and 30:1.

B. Characterization of Formulations

A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) is used to determine the particle size, the polydispersity index(PDI) and the zeta potential of the modified mRNA nanoparticles in 1×PBS in determining particle size and 15 mM PBS in determining zetapotential.

Ultraviolet-visible spectroscopy is used to determine the concentrationof modified mRNA nanoparticle formulation. After mixing, the absorbancespectrum of the solution is recorded between 230 nm and 330 nm on a DU800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea,Calif.). The modified RNA concentration in the nanoparicle formulationis calculated based on the extinction coefficient of the modified RNAused in the formulation and on the difference between the absorbance ata wavelength of 260 nm and the baseline value at a wavelength of 330 nm.

QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.)is used to evaluate the encapsulation of modified RNA by thenanoparticle. The samples are diluted, transferred to a polystyrene 96well plate, then either a TE buffer or a 2% Triton X-100 solution isadded. The plate is incubated and the RIBOGREEN® reagent is diluted inTE buffer, and of this solution is added to each well. The fluorescenceintensity is measured using a fluorescence plate reader (Wallac Victor1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.) The fluorescencevalues of the reagent blank are subtracted from each of the samples andthe percentage of free modified RNA is determined by dividing thefluorescence intensity of the intact sample by the fluorescence value ofthe disrupted sample.

C. In Vitro Incubation

Human embryonic kidney epithelial (HEK293) and hepatocellular carcinomaepithelial (HepG2) cells (LGC standards GmbH, Wesel, Germany) are seededon 96-well plates (Greiner Bio-one GmbH, Frickenhausen, Germany) andplates for HEK293 cells are precoated with collagen type1. HEK293 areseeded at a density of about 30,000 and HepG2 are seeded at a density ofabout 35,000 cells per well in 100 μl cell culture medium. Formulationscontaining mCherry mRNA (mRNA sequence shown in SEQ ID NO: 21439; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) are added directly after seeding the cells and incubated. ThemCherry cDNA with the T7 promoter, 5′ untranslated region (UTR) and 3′UTR used in in vitro transcription (IVT) is given in SEQ ID NO: 21440.

Cells are harvested by transferring the culture media supernatants to a96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg,Germany). Cells are trypsinized with ½ volume Trypsin/EDTA (Biochrom AG,Berlin, Germany), pooled with respective supernatants and fixed byadding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5%formaldehyde (Merck, Darmstadt, Germany). Samples are then submitted toa flow cytometer measurement with an excitation laser and a filter forPE-Texas Red in a LSRII cytometer (Beckton Dickinson GmbH, Heidelberg,Germany). The mean fluorescence intensity (MFI) of all events and thestandard deviation of four independent wells are presented in forsamples analyzed.

D. In Vivo Formulation Studies

Mice are administered intravenously a single dose of a formulationcontaining a modified mRNA and a reLNP. The modified mRNA administeredto the mice is selected from G-CSF (mRNA sequence shown in SEQ ID NO:21438; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1), Factor IX (mRNA shown in SEQ ID NO: 1622; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) or mCherry (mRNA sequence shown in SEQ ID NO:21439; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1).

The mice are injected with 100 ug, 10 ug or 1 ug of the formulatedmodified mRNA and are sacrificed 8 hours after they are administered theformulation. Serum from the mice administered formulations containinghuman G-CSF modified mRNA are measured by specific G-CSF ELISA and serumfrom mice administered human Factor IX modified RNA is analyzed byspecific factor IX ELISA or chromogenic assay. The liver and spleen fromthe mice administered with mCherry modified mRNA are analyzed byimmunohistochemistry (IHC) or fluorescence-activated cell sorting(FACS). As a control, a group of mice are not injected with anyformulation and their serum and tissue are collected analyzed by ELISA,FACS and/or IHC.

Example 49 In Vitro Transfection of VEGF-A

Human vascular endothelial growth factor-isoform A (VEGF-A) modifiedmRNA (mRNA sequence shown in SEQ ID NO: 1672; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) wastransfected via reverse transfection in Human Keratinocyte cells in 24multi-well plates. Human Keratinocytes cells were grown in EPILIFE®medium with Supplement S7 from Invitrogen (Carlsbad, Calif.) until theyreached a confluence of 50-70%. The cells were transfected with 0,46.875, 93.75, 187.5, 375, 750, and 1500 ng of modified mRNA (mmRNA)encoding VEGF-A which had been complexed with RNAIMAX™ from Invitrogen(Carlsbad, Calif.). The RNA:RNAIMAX™ complex was formed by firstincubating the RNA with Supplement-free EPILIFE® media in a 5×volumetric dilution for 10 minutes at room temperature. In a secondvial, RNAIMAX™ reagent was incubated with Supplement-free EPILIFE® Mediain a 10× volumetric dilution for 10 minutes at room temperature. The RNAvial was then mixed with the RNAIMAX™ vial and incubated for 20-30minutes at room temperature before being added to the cells in adrop-wise fashion.

The fully optimized mRNA encoding VEGF-A (mRNA sequence shown in SEQ IDNO: 1672; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) transfected with the Human Keratinocyte cellsincluded modifications during translation such as natural nucleosidetriphosphates (NTP), pseudouridine at each uridine site and5-methylcytosine at each cytosine site (pseudo-U/5mC), andN1-methyl-pseudouridine at each uridine site and 5-methylcytosine ateach cytosine site (N1-methyl-Pseudo-U/5mC). Cells were transfected withthe mmRNA encoding VEGF-A and secreted VEGF-A concentration (pg/ml) inthe culture medium was measured at 6, 12, 24, and 48 hourspost-transfection for each of the concentrations using an ELISA kit fromInvitrogen (Carlsbad, Calif.) following the manufacturers recommendedinstructions. These data, shown in Table 70 and FIGS. 6A, 6B and 6C,show that modified mRNA encoding VEGF-A is capable of being translatedin Human Keratinocyte cells and that VEGF-A is transported out of thecells and released into the extracellular environment.

TABLE 70 VEGF-A Dosing and Protein Secretion 6 hours 12 hours 24 hours48 hours Dose (ng) (pg/ml) (pg/ml) (pg/ml) (pg/ml) VEGF-A DoseContaining Natural NTPs 46.875 10.37 18.07 33.90 67.02 93.75 9.79 20.5441.95 65.75 187.5 14.07 24.56 45.25 64.39 375 19.16 37.53 53.61 88.28750 21.51 38.90 51.44 61.79 1500 36.11 61.90 76.70 86.54 VEGF-A DoseContaining Pseudo-U/5mC 46.875 10.13 16.67 33.99 72.88 93.75 11.00 20.0046.47 145.61 187.5 16.04 34.07 83.00 120.77 375 69.15 188.10 448.50392.44 750 133.95 304.30 524.02 526.58 1500 198.96 345.65 426.97 505.41VEGF-A Dose Containing N1-methyl-Pseudo-U/5mC 46.875 0.03 6.02 27.65100.42 93.75 12.37 46.38 121.23 167.56 187.5 104.55 365.71 1025.411056.91 375 605.89 1201.23 1653.63 1889.23 750 445.41 1036.45 1522.861954.81 1500 261.61 714.68 1053.12 1513.39

Example 50 In vivo studies of Factor IX

Human Factor IX mmRNA (Gen1; fully modified 5-methycytosine andpseudouridine) formulated in formulation buffer was delivered to micevia intramuscular injection. The results demonstrate that Factor IXprotein was elevated in serum as measured 13 hours after administration.

In this study, mice (N=5 for Factor IX, N=3 for Luciferase or Buffercontrols) were intramuscularly injected with 50 μl of the Factor IXmmRNA (mRNA sequence shown in SEQ ID NO: 1622; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),Luciferase (IVT cDNA sequence shown in SEQ ID NO: 21445; mRNA sequenceshown in SEQ ID NO: 21446, polyA tail of approximately 160 nucleotidesnot shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) or the formulation buffer (F.Buffer) at 2×100 ug/mouse.The mice were bled at 13 hours after the intramuscular injection todetermine the concentration of human the polypeptide in serum in pg/mL.The results revealed that administration of Factor IX mmRNA resulted inlevels of 1600 pg/mL at 13 hours as compared to less than 100 pg/mL ofFactor IX for either Luciferase or buffer control administration.

Example 51 Multi-Site Administration: Intramuscular and Subcutaneous

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 21438;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) modified as either Gen1 or Gen2 (5-methylcytosine (5mc) and apseudouridine (ψ) modification, G-CSF-Gen1; or N1-5-methylcytosine(N1-5mc) and a ψ modification, G-CSF-Gen2) and formulated in formulationbuffer were delivered to mice via intramuscular (IM) or subcutaneous(SC) injection. Injection of four doses or 2×50 ug (two sites) daily forthree days (24 hrs interval) was performed. The fourth dose wasadministered 6 hrs before blood collection and CBC analysis. Controlsincluded Luciferase (IVT cDNA sequence shown in SEQ ID NO: 21445; mRNAsequence shown in SEQ ID NO: 21446, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) or the formulation buffer (F.Buffer). The mice were bledat 72 hours after the first mRNA injection (6 hours after the lastmodified mRNA dose) to determine the effect of mRNA-encoded human G-CSFon the neutrophil count. The dosing regimen is shown in Table 71 as arethe resulting neutrophil counts (thousands/uL). In Table 71, anasterisk(*) indicates statistical significance at p<0.05.

For intramuscular administration, the data reveal a four fold increasein neutrophil count above control at day 3 for the Gen1 G-CSF mRNA and atwo fold increase for the Gen2 G-CSF mmRNA. For subcutaneousadministration, the data reveal a two fold increase in neutrophil countabove control at day 3 for the Gen2 G-CSF mRNA.

These data demonstrate that both 5-methylcytidine/pseudouridine and5-methylcytidine/N1-methyl-pseudouridine-modified mRNA can bebiologically active, as evidenced by specific increases in bloodneutrophil counts.

TABLE 71 Dosing Regimen Dose Dose Vol. Dosing Neutrophil Gr. TreatmentRoute N = (μg/mouse) (μl/mouse) Vehicle Thous/uL 1 G-CSF I.M 5 2 × 50 ug50 F. buffer  840* (Gen1) (four doses) 2 G-CSF S.C 5 2 × 50 ug 50 F.buffer 430 (Gen1) (four doses) 3 G-CSF I.M 5 2 × 50 ug 50 F. buffer 746* (Gen2) (four doses) 4 G-CSF S.C 5 2 × 50 ug 50 F. buffer 683(Gen2) (four doses) 5 Luc I.M. 5 2 × 50 ug 50 F. buffer 201 (Gen1) (fourdoses) 6 Luc S.C. 5 2 × 50 ug 50 F. buffer 307 (Gen1) (four doses) 7 LucI.M 5 2 × 50 ug 50 F. buffer 336 (Gen2) (four doses) 8 Luc S.C 5 2 × 50ug (four doses) 50 F. buffer 357 (Gen2) 9 F. Buffer I.M 4 0 (four doses)50 F. buffer 245 10 F. Buffer S.C. 4 0 (four doses) 50 F. buffer 509 11Untreated — 4 — 312

Example 52 Intravenous Administration

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 21438;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) modified with 5-methylcytosine (5mc) and a pseudouridine (ψ)modification (Gen 1); or having no modifications and formulated in 10%lipoplex (RNAiMax) were delivered to mice at a dose of 50 ug RNA and ina volume of 100 ul via intravenous (IV) injection at days 0, 2 and 4.Neutrophils were measured at days 1, 5 and 8. Controls includednon-specific mammalian RNA or the formulation buffer alone (F.Buffer).The mice were bled at days 1, 5 and 8 to determine the effect ofmodified mRNA-encoded human G-CSF to increase neutrophil count. Thedosing regimen is shown in Table 72 as are the resulting neutrophilcounts (thousands/uL; K/uL).

For intravenous administration, the data reveal a four to five foldincrease in neutrophil count above control at day 5 with G-CSF modifiedmRNA but not with unmodified G-CSF mRNA or non-specific controls. Bloodcount returned to baseline four days after the final injection. No otherchanges in leukocyte populations were observed.

In Table 72, an asterisk(*) indicates statistical significance atp<0.001 compared to buffer.

These data demonstrate that lipoplex-formulated5-methylcytidine/pseudouridine-modified mRNA can be biologically active,when delivered through an I.V. route of administration as evidenced byspecific increases in blood neutrophil counts. No other cell subsetswere significantly altered. Unmodified G-CSF mRNA similarly administeredshowed no pharmacologic effect on neutrophil counts.

TABLE 72 Dosing Regimen Dose Vol. (μl/ Dosing Neutrophil Gr. DayTreatment N = mouse) Vehicle K/uL 1 1 G-CSF (Gen1) 5 100 10% lipoplex2.91 2 5 G-CSF (Gen1) 5 100 10% lipoplex 5.32* 3 8 G-CSF (Gen1) 5 10010% lipoplex 2.06 4 1 G-CSF 5 100 10% lipoplex 1.88 (no modification) 55 G-CSF 5 100 10% lipoplex 1.95 (no modification) 6 8 G-CSF 5 100 10%lipoplex 2.09 (no modification) 7 1 RNA control 5 100 10% lipoplex 2.908 5 RNA control 5 100 10% lipoplex 1.68 9 8 RNA control 4 100 10%lipoplex 1.72 10 1 F. Buffer 4 100 10% lipoplex 2.51 11 5 F. Buffer 4100 10% lipoplex 1.31 12 8 F. Buffer 4 100 10% lipoplex 1.92

Example 53 Saline Formulation: Intramuscular Administration

A. Protein Expression

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 21438;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) and human EPO mmRNA (mRNA sequence shown in SEQ ID NO: 1638;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1); G-CSF modified mRNA (modified with 5-methylcytosine (5mc)and pseudouridine (ψ)) and EPO modified mRNA (modified withN1-5-methylcytosine (N1-5mc) and w modification), were formulated informulation buffer (150 mM sodium chloride, 2 mM calcium chloride, 2 mMphosphate, 0.5 mM EDTA at a pH of 6.5) and delivered to mice viaintramuscular (IM) injection at a dose of 100 ug.

Controls included Luciferase (IVT cDNA sequence shown in SEQ ID NO:21445; mRNA sequence shown in SEQ ID NO: 21446, polyA tail ofapproximately 160 nucleotides not shown in sequence, 5′ cap, Cap1, fullymodified with 5-methylcytosine at each cytosine and pseudouridinereplacement at each uridine site) or the formulation buffer (F.Buffer).The mice were bled at 13 hours after the injection to determine theconcentration of the human polypeptide in serum in pg/mL. (G-CSF groupsmeasured human G-CSF in mouse serum and EPO groups measured human EPO inmouse serum). The data are shown in Table 73.

TABLE 73 Dosing Regimen Dose Average Vol. Dosing Protein Product GroupTreatment N= (μl/mouse) Vehicle pg/mL, serum G-CSF G-CSF 5 50 Saline19.8 G-CSF Luciferase 5 50 Saline 0.5 G-CSF F. buffer 5 50 F. buffer 0.5EPO EPO 5 50 Saline 191.5 EPO Luciferase 5 50 Saline 15.0 EPO F. bufferF. buffer 4.8

B. Dose Response

Human EPO modified mRNA (mRNA sequence shown in SEQ ID NO: 1638; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) wereformulated in formulation buffer and delivered to mice via intramuscular(IM) injection.

Controls included Luciferase (mRNA sequence shown in SEQ ID NO: 21446,polyA tail of approximately 160 nucleotides not shown in sequence, 5′cap, Cap1, fully modified with 5-methylcytosine and pseudouridine) orthe formulation buffer (F.Buffer). The mice were bled at 13 hours afterthe injection to determine the concentration of the human polypeptide inserum in pg/mL. The dose and expression are shown in Table 74.

TABLE 74 Dosing Regimen and Expression Average Dose Vol. Protein ProductTreatment (μl/mouse) pg/mL, serum EPO 100 96.2 EPO 50 63.5 EPO 25 18.7EPO 10 25.9 EPO 1 2.6 Luciferase 100 0.0 F. buffer 100 1.0

Example 54 EPO Muti-Dose/Multi-Administration

Studies utilizing multiple intramuscular injection sites at one timepoint were designed and performed.

The design of a single multi-dose experiment involved using humanerythropoietin (EPO) mmRNA (mRNA sequence shown in SEQ ID NO: 1638;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) or G-CSF mmRNA (mRNA sequence shown in SEQ ID NO: 21438;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) administered in formulation buffer. The dosing vehicle (F.buffer) was used as a control. The EPO and G-CSF modified mRNA weremodified with 5-methylcytosine at each cytosine and pseudouridinereplacement at each uridine site.

Animals (n=5), Sprague-Dawley rats, were injected IM (intramuscular) forthe single unit dose of 100 ug (delivered to one thigh). Formulti-dosing 6 doses of 100 ug (delivered to two thighs) were used forboth EPO and G-CSF mmRNA. Control dosing involved use of buffer at asingle dose. Human EPO blood levels were evaluated 13 hrs postinjection.

Human EPO protein was measured in rat serum 13 hrs post intramuscularinjection. Five groups of rats were treated and evaluated. The resultsare shown in Table 75.

TABLE 75 Multi-dose study Avg. Pg/mL Dose of Total human EPO, GroupTreatment mmRNA Dose serum 1 Human EPO 1 x 100 ug 100 ug 143 mmRNA 2Human EPO 6 x 100 ug 600 ug 256 mmRNA 3 G-CSF mmRNA 1 x 100 ug 100 ug 434 G-CSF mmRNA 6 x 100 ug 600 ug 58 5 Buffer Alone — — 20

Example 55 Signal Sequence Exchange Study

Several variants of mmRNAs encoding human Granulocyte colony stimulatingfactor (G-CSF) (mRNA sequence shown in SEQ ID NO: 21438; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) weresynthesized using modified nucleotides pseudouridine and5-methylcytosine (pseudo-U/5mC). These variants included the G-CSFconstructs encoding either the wild-type N terminal secretory signalpeptide sequence (MAGPATQSPMKLMALQLLLWHSALWTVQEA; SEQ ID NO: 95), nosecretory signal peptide sequence, or secretory signal peptide sequencestaken from other mRNAs. These included sequences where the wild typeG-CSF signal peptide sequence was replaced with the signal peptidesequence of either: human α-1-anti trypsin (AAT)(MMPSSVSWGILLLAGLCCLVPVSLA; SEQ ID NO: 94), human Factor IX (FIX)(MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKR; SEQ ID NO: 96), humanProlactin (Prolac) (MKGSLLLLLVSNLLLCQSVAP; SEQ ID NO: 97), or humanAlbumin (Alb) (MKWVTFISLLFLFSSAYSRGVFRR; SEQ ID NO: 98).

250 ng of modified mRNA encoding each G-CSF variant was transfected intoHEK293A (293A in the table), mouse myoblast (MM in the table) (C2C12,CRL-1772, ATCC) and rat myoblast (RM in the table) (L6 line, CRL-1458,ATCC) cell lines in a 24 well plate using 1 ul of Lipofectamine 2000(Life Technologies), each well containing 300,000 cells. Thesupernatants were harvested after 24 hrs and the secreted G-CSF proteinwas analyzed by ELISA using the Human G-CSF ELISA kit (LifeTechnologies). The data shown in Table 76 reveal that cells transfectedwith G-CSF mmRNA encoding the Albumin signal peptide secrete at least 12fold more G-CSF protein than its wild type counterpart.

TABLE 76 Signal Peptide Exchange 293A MM RM Signal peptides (pg/ml)(pg/ml) (pg/ml) G-CSF Natural 9650 3450 6050 α-1-anti trypsin 9950 50008475 Factor IX 11675 6175 11675 Prolactin 7875 1525 9800 Albumin 12205081050 173300 No Signal peptide 0 0 0

Example 56 Cytokine Study: PBMC

A. PBMC isolation and Culture

mL of human blood from two donors was received from Research BloodComponents (lots KP30928 and KP30931) in sodium heparin tubes. For eachdonor, the blood was pooled and diluted to 70 mL with DPBS (SAFCBioscience 59331C, lot 071M8408) and split evenly between two 50 mLconical tubes. 10 mL of Ficoll Paque (GE Healthcare 17-5442-03, lot10074400) was gently dispensed below the blood layer. The tubes werecentrifuged at 2000 rpm for 30 minutes with low acceleration andbraking. The tubes were removed and the buffy coat PBMC layers weregently transferred to a fresh 50 mL conical and washed with DPBS. Thetubes were centrifuged at 1450 rpm for 10 minutes.

The supernatant was aspirated and the PBMC pellets were resuspended andwashed in 50 mL of DPBS. The tubes were centrifuged at 1250 rpm for 10minutes. This wash step was repeated, and the PBMC pellets wereresuspended in 19 mL of Optimem I (Gibco 11058, lot 1072088) andcounted. The cell suspensions were adjusted to a concentration of3.0×10̂6 cells/mL live cells.

These cells were then plated on five 96 well tissue culture treatedround bottom plates (Costar 3799) per donor at 50 uL per well. Within 30minutes, transfection mixtures were added to each well at a volume of 50uL per well. After 4 hours post transfection, the media was supplementedwith 10 uL of Fetal Bovine Serum (Gibco 10082, lot 1012368)

B. Transfection Preparation

mmRNA encoding human G-CSF (mRNA sequence shown in SEQ ID NO: 21438;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) (containing either (1) natural NTPs, (2) 100% substitutionwith 5-methyl cytidine and pseudouridine, or (3) 100% substitution with5-methyl cytidine and N1-methyl-pseudouridine; mmRNA encoding luciferase(IVT cDNA sequence shown in SEQ ID NO: 21445; mRNA sequence shown in SEQID NO: 21446, polyA tail of approximately 160 nucleotides not shown insequence, 5′ cap, Cap1, fully modified with 5-methylcytosine at eachcytosine and pseudouridine replacement at each uridine site) (containingeither (1) natural NTPs or (2) 100% substitution with 5-methyl cytidineand pseudouridine) and TLR agonist R848 (Invivogen tlr1-r848) werediluted to 38.4 ng/uL in a final volume of 2500 uL Optimem I.

Separately, 432 uL of Lipofectamine 2000 (Invitrogen 11668-027, lot1070962) was diluted with 13.1 mL Optimem I. In a 96 well plate ninealiquots of 135 uL of each mmRNA, positive control (R-848) or negativecontrol (Optimem I) was added to 135 uL of the diluted Lipofectamine2000. The plate containing the material to be transfected was incubatedfor 20 minutes. The transfection mixtures were then transferred to eachof the human PBMC plates at 50 uL per well. The plates were thenincubated at 37C. At 2, 4, 8, 20, and 44 hours each plate was removedfrom the incubator, and the supernatants were frozen.

After the last plate was removed, the supernatants were assayed using ahuman G-CSF ELISA kit (Invitrogen KHC2032) and human IFN-alpha ELISA kit(Thermo Scientific 41105-2). Each condition was done in duplicate.

C. Results

The ability of unmodified and modified mRNA (mmRNAs) to produce theencoded protein was assessed (G-CSF production) over time as was theability of the mRNA to trigger innate immune recognition as measured byinterferon-alpha production. Use of in vitro PBMC cultures is anaccepted way to measure the immunostimulatory potential ofoligonucleotides (Robbins et al., Oligonucleotides 2009 19:89-102;herein incorporated by reference in its entirety).

Results were interpolated against the standard curve of each ELISA plateusing a four parameter logistic curve fit. Shown in Tables 77 and 78 arethe average from 2 separate PBMC donors of the G-CSF and IFN-alphaproduction over time as measured by specific ELISA.

In the G-CSF ELISA, background signal from the Lipofectamine 2000untreated condition was subtracted at each timepoint. The datademonstrated specific production of human G-CSF protein by humanperipheral blood mononuclear is seen with G-CSF mRNA containing naturalNTPs, 100% substitution with 5-methyl cytidine and pseudouridine, or100% substitution with 5-methyl cytidine and N1-methyl-pseudouridine.Production of G-CSF was significantly increased through the use ofmodified mRNA relative to unmodified mRNA, with the 5-methyl cytidineand N1-methyl-pseudouridine containing G-CSF mmRNA showing the highestlevel of G-CSF production. With regards to innate immune recognition,unmodified mRNA resulted in substantial IFN-alpha production, while themodified mRNA largely prevented interferon-alpha production. G-CSF mRNAfully modified with 5-methyl cytidine and N1-methyl-pseudouridine didnot significantly increase cytokines whereas G-CSF mRNA fully modifiedwith 5-methyl cytidine and pseudouridine induced IFN-alpha, TNF-alphaand IP10. Many other cytokines were not affected by either modification.

TABLE 77 G-CSF Signal G-CSF signal - 2 Donor Average pg/mL 2 Hr 4 Hr 8Hr 20 Hr 44 Hr G-CSF (5mC/pseudouridine) 120.3 136.8 421.0 346.1 431.8G-CSF (5mC/N1-methyl 256.3 273.7 919.3 1603.3 1843.3 pseudouridine)G-CSF(Natural-no modification) 63.5 92.6 129.6 258.3 242.4 Luciferase(5mC/pseudouridine) 4.5 153.7 33.0 186.5 58.0

TABLE 78 IFN-alpha signal IFN-alpha signal - 2 donor average pg/mL 2 Hr4 Hr 8 Hr 20 Hr 44 Hr G-CSF (5mC/pseudouridine) 21.1 2.9 3.7 22.7 4.3G-CSF (5mC/N1-methyl- 0.5 0.4 3.0 2.3 2.1 pseudouridine) G-CSF(Natural)0.0 2.1 23.3 74.9 119.7 Luciferase (5mC/pseudouridine) 0.4 0.4 4.7 1.02.4 R-848 39.1 151.3 278.4 362.2 208.1 Lpf. 2000 control 0.8 17.2 16.50.7 3.1

Example 57 Chemical modification ranges of modified mRNA

Modified nucleotides such as, but not limited to, the chemicalmodifications 5-methylcytosine and pseudouridine have been shown tolower the innate immune response and increase expression of RNA inmammalian cells. Surprisingly, and not previously known, the effectsmanifested by the chemical modifications can be titrated when the amountof chemical modification is less than 100%. Previously, it was believedthat full modification was necessary and sufficient to elicit thebeneficial effects of the chemical modifications and that less than 100%modification of an mRNA had little effect. However, it has now beenshown that the benefits of chemical modification can be derived usingless than complete modification and that the effects are target,concentration and modification dependent.

A. Modified RNA transfected in PBMC

960 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) andpseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with0.8 uL of Lipofectamine 2000 into peripheral blood mononuclear cells(PBMC) from three normal blood donors (D1, D2, D3). The G-CSF mRNA (mRNAsequence shown in SEQ ID NO: 21438; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) was completely modifiedwith 5mC and pseudoU (100% modification), not modified with 5mC andpseudoU (0% modification) or was partially modified with 5mC and pseudoUso the mRNA would contain 50% modification, 25% modification, 10%modification, %5 modification, 1% modification or 0.1% modification. Acontrol sample of Luciferase (mRNA sequence shown in SEQ ID NO: 21446;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified 5meC and pseudoU) was also analyzed for G-CSFexpression. For TNF-alpha and IFN-alpha control samples ofLipofectamine-2000, LPS, R-848, Luciferase (mRNA sequence shown in SEQID NO: 21446; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified 5mC and pseudo), and P(I)P(C)were also analyzed. The supernatant was harvested and run by ELISA 22hours after transfection to determine the protein expression. Theexpression of G-CSF is shown in Table 79 and the expression of IFN-alphaand TNF-alpha is shown in Table 80. The expression of IFN-alpha andTNF-alpha may be a secondary effect from the transfection of the G-CSFmRNA. Tables 79 and 80 show that the amount of chemical modification ofG-CSF, IFN-alpha and TNF-alpha is titratable when the mRNA is not fullymodified and the titratable trend is not the same for each target.

TABLE 79 G-CSF Expression G-CSF Expression (pg/ml) D1 D2 D3 100%modification 270.3 151.6 162.2 50% modification 45.6 19.8 26.3 25%modification 23.6 10.8 8.9 10% modification 39.4 12.9 12.9 5%modification 70.9 26.8 26.3 1% modification 70.3 26.9 66.9 0.1%modification 67.5 25.2 28.7 Luciferase 14.5 3.1 10.0

TABLE 80 IFN-alpha and TNF-alpha Expression IFN-alpha ExpressionTNF-alpha Expression (pg/ml) (pg/ml) D1 D2 D3 D1 D2 D3 100% 76.8 6.815.1 5.6 1.4 21.4 modification 50% 22.0 5.5 257.3 4.7 1.7 12.1modification 25% 64.1 14.9 549.7 3.9 0.7 10.1 modification 10% 150.218.8 787.8 6.6 0.9 13.4 modification 5% modification 143.9 41.3 1009.62.5 1.8 12.0 1 % 189.1 40.5 375.2 9.1 1.2 25.7 modification 0.1% 261.237.8 392.8 9.0 2. 13.7 modification 0% modification 230.3 45.1 558.310.9 1.4 10.9 LF 200 0 0 1.5 45.8 2.8 53.6 LPS 0 0 1.0 114.5 70.0 227.0R-848 39.5 11.9 183.5 389.3 256.6 410.6 Luciferase 9.1 0 3.9 4.5 2.713.6 P(I)P(C) 1498.1 216.8 238.8 61.2 4.4 69.1

B. Modified RNA transfected in HEK293

Human embryonic kidney epithelial (HEK293) cells were seeded on 96-wellplates at a density of 30,000 cells per well in 100 ul cell culturemedium. 250 ng of modified G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) formulated with RNAiMAX™ (Invitrogen, Carlsbad,Calif.) was added to a well. The G-CSF was completely modified with 5mCand pseudoU (100% modification), not modified with 5mC and pseudoU (0%modification) or was partially modified with 5mC and pseudoU so the mRNAwould contain 75% modification, 50% modification or 25% modification.Control samples (AK 5/2, mCherry (SEQ ID NO: 21439; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified 5mC and pseudoU) and untreated) were also analyzed. Thehalf-life of G-CSF mRNA fully modified with 5-methylcytosine andpseudouridine is approximately 8-10 hours. The supernatants wereharvested after 16 hours and the secreted G-CSF protein was analyzed byELISA. Table 81 shows that the amount of chemical modification of G-CSFis titratable when the mRNA is not fully modified.

TABLE 81 G-CSF Expression G-CSF Expression (ng/ml) 100% modification118.4 75% modification 101.9 50% modification 105.7 25% modification231.1 0% modification 270.9 AK 5/2 166.8 mCherry 0 Untreated 0

Example 58 In Vivo Delivery of Modified mRNA (mmRNA)

Modified RNA was delivered to C57/BL6 mice intramuscularly,subcutaneously, or intravenously to evaluate the bio-distribution ofmodified RNA using luciferase. A formulation buffer used with alldelivery methods contained 150 mM sodium chloride, 2 mM calciumchloride, 2 mM Na⁺-phosphate which included 1.4 mM monobasic sodiumphosphate and 0.6 mM of dibasic sodium phosphate, and 0.5 mMethylenediaminetetraacetic acid (EDTA) was adjusted using sodiumhydroxide to reach a final pH of 6.5 before being filtered andsterilized. A 1× concentration was used as the delivery buffer. Tocreate the lipoplexed solution delivered to the mice, in one vial 50 μgof RNA was equilibrated for 10 minutes at room temperature in thedelivery buffer and in a second vial 10 μl RNAiMAX™ was equilibrated for10 minutes at room temperature in the delivery buffer. Afterequilibrium, the vials were combined and delivery buffer was added toreach a final volume of 100 μl which was then incubated for 20 minutesat room temperature. Luciferin was administered by intraperitonealinjection (IP) at 150 mg/kg to each mouse prior to imaging during theplateau phase of the luciferin exposure curve which was between 15 and30 minutes. To create luciferin, 1 g of D-luciferin potassium or sodiumsalt was dissolved in 66.6 ml of distilled phosphate buffer solution(DPBS), not containing Mg2+ or Ca2+, to make a 15 mg/ml solution. Thesolution was gently mixed and passed through a 0.2 μm syringe filter,before being purged with nitrogen, aliquoted and frozen at −80° C. whilebeing protected from light as much as possible. The solution was thawedusing a waterbath if luciferin was not dissolved, gently mixed and kepton ice on the day of dosing.

Whole body images were taken of each mouse 2, 8 and 24 hours afterdosing. Tissue images and serum was collected from each mouse 24 hoursafter dosing. Mice administered doses intravenously had their liver,spleen, kidneys, lungs, heart, peri-renal adipose tissue and thymusimaged. Mice administered doses intramuscularly or subcutaneously hadtheir liver, spleen, kidneys, lungs, peri-renal adipose tissue, andmuscle at the injection site. From the whole body images thebioluminescence was measured in photon per second for each route ofadministration and dosing regimen.

A. Intramuscular Administration

Mice were intramuscularly (I.M.) administered either modified luciferasemRNA fully modified with 5-methylcytosine and pseudouridine (Naked-Luc),lipoplexed modified luciferase mRNA fully modified with 5-methylcytosineand pseudouridine (Lipoplex-luc) (IVT cDNA sequence shown in SEQ ID NO:21445; mRNA sequence shown in SEQ ID NO: 21446, polyA tail ofapproximately 160 nucleotides not shown in sequence, 5′ cap, Cap1, fullymodified with 5-methylcytosine at each cytosine and pseudouridinereplacement at each uridine site), lipoplexed modified granulocytecolony-stimulating factor (G-CSF) mRNA (mRNA sequence shown in SEQ IDNO: 21438; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) (Lipoplex-Cytokine) or the formation buffer at a singledose of 50 μl of modified RNA in an injection volume of 50 μl for eachformulation in the right hind limb and a single dose of 5 μg of modifiedRNA in an injection volume of 50 μl in the left hind limb. Thebioluminescence average for the luciferase expression signals for eachgroup at 2, 8 and 24 hours after dosing are shown in Table 82. Thebioluminescence showed a positive signal at the injection site of the 5μg and 50 μg modified RNA formulations containing and not containinglipoplex.

TABLE 82 In vivo Biophotoic Imaging (I.M. Injection Route) DoseBioluminescence (photon/sec) Formulation (ug) 2 hours 8 hours 24 hoursNaked-Luc 5 224,000 683,000 927,000 Lipolplex-Luc 5 579,000 639,000186,000 Lipoplex-G-CSF 5 64,600 85,600 75,100 Formulation Buffer 5102,000 86,000 90,700 Naked-Luc 50 446,000 766,000 509,000 Lipolplex-Luc50 374,000 501,000 332,000 Lipoplex-G-CSF 50 49,400 74,800 74,200Formulation Buffer 50 59,300 69,200 63,600

B. Subcutaneous Administration

Mice were subcutaneously (S.C.) administered either modified luciferasemRNA (Naked-Luc), lipoplexed modified luciferase mRNA (Lipoplex-luc),lipoplexed modified G-CSF mRNA (Lipoplex-G-CSF) or the formation bufferat a single dose of 50 μg of modified mRNA in an injection volume of 100μl for each formulation. The bioluminescence average for the luciferaseexpression signals for each group at 2, 8 and 24 hours after dosing areshown in Table 83. The bioluminescence showed a positive signal at theinjection site of the 50 μg modified mRNA formulations containing andnot containing lipoplex.

TABLE 83 In vivo Biophotoic Imaging (S.C. Injection Route)Bioluminescence (photon/sec) Formulation 2 hours 8 hours 24 hoursNaked-Luc 3,700,000 8,060,000 2,080,000 Lipolplex-Luc 3,960,0001,700,000 1,290,000 Lipoplex-G-CSF 123,000 121,000 117,000 FormulationBuffer 116,000 127,000 123,000

C. Intravenous Administration

Mice were intravenously (I.V.) administered either modified luciferasemRNA (Naked-Luc), lipoplexed modified luciferase mRNA (Lipoplex-luc),lipoplexed modified G-CSF mRNA (Lipoplex-G-CSF) or the formation bufferat a single dose of 50 μg of modified mRNA in an injection volume of 100μl for each formulation. The bioluminescence average for the luciferaseexpression signal in the spleen from each group at 2 hours after dosingis shown in Table 84. The bioluminescence showed a positive signal inthe spleen of the 50 μg modified mRNA formulations containing lipoplex.

TABLE 84 In vivo Biophotoic Imaging (I.V. Injection Route)Bioluminescence (photon/sec) Formulation of the Spleen Naked-Luc 58,400Lipolplex-Luc 65,000 Lipoplex-G-CSF 57,100 Formulation Buffer 58,300

Example 59 Buffer Formulation Studies

G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 21438; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1;fully modified with N1-pseudouridine and 5-methylcytosine) or Factor IXmodified mRNA (mRNA sequence shown in SEQ ID NO: 1622; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with N1-pseudouridine and 5-methylcytosine) in a buffersolution is administered intramuscularly to rats in an injection volumeof 50 μl (n=5) at a modified mRNA dose of 200 ug per rat as described inTable 85. The modified mRNA is lyophilized in water for 1-2 days. It isthen reconstituted in the buffers listed below to a target concentrationof 6 mg/ml. Concentration is determined by OD 260. Samples are dilutedto 4 mg/ml in the appropriate buffer before dosing.

To precipitate the modified mRNA, 3M sodium acetate, pH 5.5 and pureethanol are added at 1/10^(th) the total volume and 4 times the totalvolume of modified mRNA, respectively. The material is placed at −80 Cfor a minimum of 1 hour. The material is then centrifuged for 30 minutesat 4000 rpm, 4 C. The supernatant is removed and the pellet iscentrifuged and washed 3× with 75% ethanol. Finally, the pellet isreconstituted with buffer to a target concentration of 6 mg/ml.Concentration is determined by OD 260. Samples are diluted to 4 mg/ml inthe appropriate buffer before dosing. All samples are prepared bylyophilization unless noted below.

TABLE 85 Buffer Dosing Groups Dose Group Treatment Buffer (ug/rat) 1G-CSF 0.9% Saline 200 Factor IX 0.9% Saline 200 2 G-CSF 0.9% Saline + 2mM 200 Calcium Factor IX 0.9% Saline + 2 mM 200 Calcium 3 G-CSF LactatedRinger's 200 Factor IX Lactated Ringer's 200 4 G-CSF 5% Sucrose 200Factor IX 5% Sucrose 200 5 G-CSF 5% Sucrose + 2 mM 200 Calcium Factor IX5% Sucrose + 2 mM 200 Calcium 6 G-CSF 5% Mannitol 200 Factor IX 5%Mannitol 200 7 G-CSF 5% Mannitol + 2 mM 200 Calcium Factor IX 5%Mannitol + 2 mM 200 Calcium 8 G-CSF 0.9% saline (precipitation) 200Factor IX 0.9% saline (precipitation) 200

Serum samples are collected from the rats at various time intervals andanalyzed for G-CSF or Factor IX protein expression using G-CSF or FactorIX ELISA.

Example 60 Multi-Dose Study

Sprague-Dawley rats (n=8) are injected intravenously eight times (twicea week) over 28 days. The rats are injected with 0.5 mg/kg, 0.05 mg/kg,0.005 mg/kg or 0.0005 mg/kg of human G-CSF modified mRNA of luciferasemodified mRNA formulated in a lipid nanoparticle, 0.5 mg/kg of humanG-CSF modified mRNA in saline, 0.2 mg/kg of the human G-CSF proteinNeupogen or non-translatable human G-CSF modified mRNA formulated in alipid nanoparticle. Serum is collected during pre-determined timeintervals to evaluate G-CSF protein expression (8, 24 and 72 hours afterthe first dose of the week), complete blood count and white blood count(24 and 72 hours after the first dose of the week) and clinicalchemistry (24 and 72 hours after the first dose of the week). The ratsare sacrificed at day 29, 4 days after the final dosing, to determinethe complete blood count, white blood count, clinical chemistry, proteinexpression and to evaluate the effect on the major organs byhistopathology and necropsy. Further, an antibody assay is performed onthe rats on day 29.

Example 61 LNP In Vivo Study

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyAtail of approximately 160 nucleotides not shown in sequence, 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine wasformulated as a lipid nanoparticle (LNP) using the syringe pump method.The LNP was formulated at a 20:1 weight ratio of total lipid to modifiedmRNA with a final lipid molar ratio of 50:10:38.5:1.5 (DLin-KC2-DMA:DSPC: Cholesterol: PEG-DMG). As shown in Table 86, the luciferase LNPformulation was characterized by particle size, zeta potential, andencapsulation.

TABLE 86 Luciferase Formulation Formulation NPA-098-1 Modified mRNALuciferase Mean size 135 nm PDI: 0.08 Zeta at pH 7.4 −0.6 mV Encaps.(RiboGr) 91%

As outlined in Table 87, the luciferase LNP formulation was administeredto Balb-C mice (n=3) intramuscularly, intravenously and subcutaneouslyand a luciferase modified RNA formulated in PBS was administered to miceintravenously.

TABLE 87 Luciferase Formulations Amount of Concen- Injection modifiedtration Volume RNA Dose Formulation Vehicle Route (mg/ml) (ul) (ug)(mg/kg) Luc-LNP PBS IV 0.20 50 10 0.50 Luc-LNP PBS IM 0.20 50 10 0.50Luc-LNP PBS SC 0.20 50 10 0.50 Luc-PBS PBS IV 0.20 50 10 0.50

The mice administered the luciferase LNP formulation intravenously andintramuscularly were imaged at 2, 8, 24, 48, 120 and 192 hours and themice administered the luciferase LNP formulation subcutaneously wereimaged at 2, 8, 24, 48 and 120 hours to determine the luciferaseexpression as shown in Table 88. In Table 88, “NT” means not tested.Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse.

TABLE 88 Luciferase Expression Route of Average Expression(photon/second) Form. Administration 2 hours 8 hours 24 hours 48 hours120 hours 192 hours Luc- IV 1.62E+08 3.00E+09 7.77E+08 4.98E+08 1.89E+086.08E+07 LNP Luc- IM 4.85E+07 4.92E+08 9.02E+07 3.17E+07 1.22E+072.38E+06 LNP Luc- SC 1.85E+07 9.79E+08 3.09E+08 4.94E+07 1.98E+06 NT LNPLuc- IV 3.61E+05 5.64E+05 3.19E+05 NT NT NT PBS

One mouse administered the LNP formulation intravenously was sacrificedat 8 hours to determine the luciferase expression in the liver andspleen. Also, one mouse administered the LNP formulation intramuscularwas sacrificed at 8 hours to determine the luciferase expression of themuscle around the injection site and in the liver and spleen. As shownin Table 89, expression was seen in the both the liver and spleen afterintravenous and intramuscular administration and in the muscle aroundthe intramuscular injection site.

TABLE 89 Luciferase Expression in Tissue Expression (photon/second)Luciferase LNP: IV Administration Liver 7.984E+08 Spleen 3.951E+08Luciferase LNP: IM Administration Muscle around the injection site3.688E+07 Liver 1.507E+08 Spleen 1.096E+07

Example 62 Cytokine Study: PBMC

A. PBMC isolation and Culture

mL of human blood from two donors was received from Research BloodComponents (lots KP30928 and KP30931) in sodium heparin tubes. For eachdonor, the blood was pooled and diluted to 70 mL with DPBS (SAFCBioscience 59331 C, lot 071M8408) and split evenly between two 50 mLconical tubes. 10 mL of Ficoll Paque (GE Healthcare 17-5442-03, lot10074400) was gently dispensed below the blood layer. The tubes werecentrifuged at 2000 rpm for 30 minutes with low acceleration and brakingThe tubes were removed and the buffy coat PBMC layers were gentlytransferred to a fresh 50 mL conical and washed with DPBS. The tubeswere centrifuged at 1450 rpm for 10 minutes.

The supernatant was aspirated and the PBMC pellets were resuspended andwashed in 50 mL of DPBS. The tubes were centrifuged at 1250 rpm for 10minutes. This wash step was repeated, and the PBMC pellets wereresuspended in 19 mL of Optimem I (Gibco 11058, lot 1072088) andcounted. The cell suspensions were adjusted to a concentration of3.0×10̂6 cells/mL live cells.

These cells were then plated on five 96 well tissue culture treatedround bottom plates (Costar 3799) per donor at 50 uL per well. Within 30minutes, transfection mixtures were added to each well at a volume of 50uL per well. After 4 hours post transfection, the media was supplementedwith 10 uL of Fetal Bovine Serum (Gibco 10082, lot 1012368).

B. Transfection Preparation

Modified mRNA encoding human G-CSF (mRNA sequence shown in SEQ ID NO:21438; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) (containing either (1) natural NTPs, (2) 100%substitution with 5-methyl cytidine and pseudouridine, or (3) 100%substitution with 5-methyl cytidine and N1-methyl-pseudouridine; mRNAencoding luciferase (IVT cDNA sequence shown in SEQ ID NO: 21445; mRNAsequence shown in SEQ ID NO: 21446, polyA tail of approximately 160nucleotides not shown in sequence, 5′ cap, Cap1, fully modified with5-methylcytosine at each cytosine and pseudouridine replacement at eachuridine site) (containing either (1) natural NTPs or (2) 100%substitution with 5-methyl cytidine and pseudouridine) and TLR agonistR848 (Invivogen tlr1-r848) were diluted to 38.4 ng/uL in a final volumeof 2500 uL Optimem I.

Separately, 110 uL of Lipofectamine 2000 (Invitrogen 11668-027, lot1070962) was diluted with 6.76 mL Optimem I. In a 96 well plate ninealiquots of 135 uL of each mRNA, positive control (R-848) or negativecontrol (Optimem I) was added to 135 uL of the diluted Lipofectamine2000. The plate containing the material to be transfected was incubatedfor 20 minutes. The transfection mixtures were then transferred to eachof the human PBMC plates at 50 uL per well. The plates were thenincubated at 37° C. At 2, 4, 8, 20, and 44 hours each plate was removedfrom the incubator, and the supernatants were frozen.

After the last plate was removed, the supernatants were assayed using ahuman G-CSF ELISA kit (Invitrogen KHC2032) and human IFN-alpha ELISA kit(Thermo Scientific 41105-2). Each condition was done in duplicate.

C. Protein and Innate Immune Response Analysis

The ability of unmodified and modified mRNA to produce the encodedprotein was assessed (G-CSF production) over time as was the ability ofthe mRNA to trigger innate immune recognition as measured byinterferon-alpha production. Use of in vitro PBMC cultures is anaccepted way to measure the immunostimulatory potential ofoligonucleotides (Robbins et al., Oligonucleotides 2009 19:89-102).

Results were interpolated against the standard curve of each ELISA plateusing a four parameter logistic curve fit. Shown in Tables 90 and 91 arethe average from 3 separate PBMC donors of the G-CSF, interferon-alpha(IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) production overtime as measured by specific ELISA.

In the G-CSF ELISA, background signal from the Lipofectamine 2000(LF2000) untreated condition was subtracted at each time point. The datademonstrated specific production of human G-CSF protein by humanperipheral blood mononuclear is seen with G-CSF mRNA containing naturalNTPs, 100% substitution with 5-methyl cytidine and pseudouridine, or100% substitution with 5-methyl cytidine and N1-methyl-pseudouridine.Production of G-CSF was significantly increased through the use of5-methyl cytidine and N1-methyl-pseudouridine modified mRNA relative to5-methyl cytidine and pseudouridine modified mRNA.

With regards to innate immune recognition, while both modified mRNAchemistries largely prevented IFN-alpha and TNF-alpha productionrelative to positive controls (R848, p(I)p(C)), significant differencesdid exist between the chemistries. 5-methyl cytidine and pseudouridinemodified mRNA resulted in low but detectable levels of IFN-alpha andTNF-alpha production, while 5-methyl cytidine andN1-methyl-pseudouridine modified mRNA resulted in no detectableIFN-alpha and TNF-alpha production.

Consequently, it has been determined that, in addition to the need toreview more than one cytokine marker of the activation of the innateimmune response, it has surprisingly been found that combinations ofmodifications provide differing levels of cellular response (proteinproduction and immune activation). The modification,N1-methyl-pseudouridine, in this study has been shown to convey addedprotection over the standard combination of5-methylcytidine/pseudouridine explored by others resulting in twice asmuch protein and almost 150 fold reduction in immune activation(TNF-alpha).

Given that PBMC contain a large array of innate immune RNA recognitionsensors and are also capable of protein translation, it offers a usefulsystem to test the interdependency of these two pathways. It is knownthat mRNA translation can be negatively affected by activation of suchinnate immune pathways (Kariko et al. Immunity (2005) 23:165-175; Warrenet al. Cell Stem Cell (2010) 7:618-630). Using PBMC as an in vitro assaysystem it is possible to establish a correlation between translation (inthis case G-CSF protein production) and cytokine production (in thiscase exemplified by IFN-alpha and TNF-alpha protein production). Betterprotein production is correlated with lower induction of innate immuneactivation pathway, and new chemistries can be judged favorably based onthis ratio (Table 92).

In this study, the PC Ratio for the two chemical modifications,pseudouridine and N1-methyl-pseudouridine, both with 5-methy cytosinewas 4742/141=34 as compared to 9944/1=9944 for the cytokine IFN-alpha.For the cytokine, TNF-alpha, the two chemistries had PC Ratios of 153and 1243, respectively suggesting that for either cytokine, theN1-methyl-pseudouridine is the superior modification. In Tables 90 and91, “NT” means not tested.

TABLE 90 G-CSF G-CSF: 3 Donor Average (pg/ml) G-CSF 47425-methylcytosine/pseudouridine G-CSF 99445-methylcytosine/N1-methyl-pseudouridine Luciferase 18 LF2000 16

TABLE 91 IFN-alpha and TNF-alpha IFN-alpha: TNF-alpha: 3 Donor Average 3Donor Average (pg/ml) (pg/ml) G-CSF 141 31 5-methylcytosine/pseudouridine G-CSF 1 8 5-methylcytosine/ N1-methyl-pseudouridineP(I)P(C) 1104 NT R-848 NT 1477 LF2000 17 25

TABLE 92 G-CSF to Cytokine Ratios G-CSF/IFN-alpha (ratio)G-CSF/TNF-alpha (ratio) 5-methyl 5-methylcytosine/ 5-methyl5-methylcytosine/ cytosine/ N1-methyl- cytosine/ N1-methyl-pseudouridine pseudouridine pseudouridine pseudouridine PC Ratio 34 9944153 1243

Example 63 In Vitro PBMC Studies: Percent modification

480 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) andpseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with0.4 uL of Lipofectamine 2000 into peripheral blood mononuclear cells(PBMC) from three normal blood donors (D1, D2, and D3). The G-CSF mRNA(mRNA sequence shown in SEQ ID NO: 21438; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap1) was completelymodified with 5mC and pseudoU (100% modification), not modified with 5mCand pseudoU (0% modification) or was partially modified with 5mC andpseudoU so the mRNA would contain 75% modification, 50% modification or25% modification. A control sample of Luciferase (mRNA sequence shown inSEQ ID NO: 21446; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1; fully modified 5meC and pseudoU) was alsoanalyzed for G-CSF expression. For TNF-alpha and IFN-alpha controlsamples of Lipofectamine-2000, LPS, R-848, Luciferase (mRNA sequenceshown in SEQ ID NO: 21446; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified 5mC and pseudo), andP(I)P(C) were also analyzed. The supernatant was harvested and run byELISA 22 hours after transfection to determine the protein expression.The expression of G-CSF is shown in Table 93 and the expression ofIFN-alpha and TNF-alpha is shown in Table 94. The expression ofIFN-alpha and TNF-alpha may be a secondary effect from the transfectionof the G-CSF mRNA. Tables 93 and 94 show that the amount of chemicalmodification of G-CSF, interferon alpha (IFN-alpha) and tumor necrosisfactor-alpha (TNF-alpha) is titratable when the mRNA is not fullymodified and the titratable trend is not the same for each target.

By using PBMC as an in vitro assay system it is possible to establish acorrelation between translation (in this case G-CSF protein production)and cytokine production (in this case exemplified by IFN-alpha proteinproduction). Better protein production is correlated with lowerinduction of innate immune activation pathway, and the percentagemodification of a chemistry can be judged favorably based on this ratio(Table 95). As calculated from Tables 93 and 94 and shown in Table 95,full modification with 5-methylcytidine and pseudouridine shows a muchbetter ratio of protein cytokine production than without anymodification (natural G-CSF mRNA) (100-fold for IFN-alpha and 27-foldfor TNF-alpha). Partial modification shows a linear relationship withincreasingly less modification resulting in a lower protein cytokineratio.

TABLE 93 G-CSF Expression G-CSF Expression (pg/ml) D1 D2 D3 100%modification  1968.9 2595.6 2835.7 75% modification 566.7 631.4 659.550% modification 188.9 187.2 191.9 25% modification 139.3 126.9 102.0 0% modification 194.8 182.0 183.3 Luciferase 90.2 0.0 22.1

TABLE 94 IFN-alpha and TNF-alpha Expression IFN-alpha Expression (pg/ml)TNF-alpha Expression (pg/ml) D1 D2 D3 D1 D2 D3 100% modification 336.578.0 46.4 115.0 15.0 11.1  75% modification 339.6 107.6 160.9 107.4 21.711.8  50% modification 478.9 261.1 389.7 49.6 24.1 10.4  25%modification 564.3 400.4 670.7 85.6 26.6 19.8  0% modification 1421.6810.5 1260.5 154.6 96.8 45.9 LPS 0.0 0.6 0.0 0.0 12.6 4.3 R-848 0.5 3.014.1 655.2 989.9 420.4 P(I)P(C) 130.8 297.1 585.2 765.8 2362.7 1874.4Lipid only 1952.2 866.6 855.8 248.5 82.0 60.7

TABLE 95 PC Ratio and Effect of Percentage of Modification AverageAverage Average G-CSF/IFN- G-CSF/TNF- % G-CSF IFN-a TNF-a alpha alphaModification (pg/ml) (pg/ml) (pg/ml) (PC ratio) (PC ratio) 100 2466 15347 16 52 75 619 202 47 3.1 13 50 189 376 28 0.5 6.8 25 122 545 44 0.22.8 0 186 1164 99 0.16 1.9

Example 64 Modified RNA transfected in PBMC

500 ng of G-CSF mRNA modified with 5-methylcytosine (5mC) andpseudouridine (pseudoU) or unmodified G-CSF mRNA was transfected with0.4 uL of Lipofectamine 2000 into peripheral blood mononuclear cells(PBMC) from three normal blood donors (D1, D2, and D3). The G-CSF mRNA(mRNA sequence shown in SEQ ID NO: 21438; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap1) was completelymodified with 5mC and pseudoU (100% modification), not modified with 5mCand pseudoU (0% modification) or was partially modified with 5mC andpseudoU so the mRNA would contain 50% modification, 25% modification,10% modification, %5 modification, 1% modification or 0.1% modification.A control sample of mCherry (mRNA sequence shown in SEQ ID NO: 21439;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified 5meC and pseudouridine), G-CSF fully modifiedwith 5-methylcytosine and pseudouridine (Control G-CSF) and an untreatedcontrol was also analyzed for expression of G-CSF, tumor necrosisfactor-alpha (TNF-alpha) and interferon-alpha (IFN-alpha). Thesupernatant was harvested 6 hours and 18 hours after transfection andrun by ELISA to determine the protein expression. The expression ofG-CSF, IFN-alpha, and TNF-alpha for Donor 1 is shown in Table 96, Donor2 is shown in Table 97 and Donor 3 is shown in Table 98.

Full 100% modification with 5-methylcytidine and pseudouridine resultedin the most protein translation (G-CSF) and the least amount of cytokineproduced across all three human PBMC donors. Decreasing amounts ofmodification results in more cytokine production (IFN-alpha andTNF-alpha), thus further highlighting the importance of fullymodification to reduce cytokines and to improve protein translation (asevidenced here by G-CSF production).

TABLE 96 Donor 1 G-CSF IFN- TNF- (pg/mL) alpha (pg/mL) alpha(pg/mL) 6hours 18 hours 6 hours 18 hours 6 hours 18 hours 100% Mod 1815 2224 1 130 0  75% Mod 591 614 0 89 0 0  50% Mod 172 147 0 193 0 0  25% Mod 111 922 219 0 0  10% Mod 138 138 7 536 18 0  1% Mod 199 214 9 660 18 3  0.1%Mod 222 208 10 597 0 6  0% Mod 273 299 10 501 10 0 Control 957 1274 3123 18633 1620 G-CSF mCherry 0 0 0 10 0 0 Untreated N/A N/A 0 0 1 1

TABLE 97 Donor 2 G-CSF IFN- TNF- (pg/mL) alpha (pg/mL) alpha (pg/mL) 6hours 18 hours 6 hours 18 hours 6 hours 18 hours 100% Mod 2184 2432 0 70 11  75% Mod 935 958 3 130 0 0  50% Mod 192 253 2 625 7 23  25% Mod 153158 7 464 6 6  10% Mod 203 223 25 700 22 39  1% Mod 288 275 27 962 51 66 0.1% Mod 318 288 33 635 28 5  0% Mod 389 413 26 748 1 253 Control 14611634 1 59 481 814 G-CSF mCherry 0 7 0 1 0 0 Untreated N/A N/A 1 0 0 0

TABLE 98 Donor 3 G-CSF IFN- TNF- (pg/mL) alpha (pg/mL) alpha(pg/mL) 6hours 18 hours 6 hours 18 hours 6 hours 18 hours 100% Mod 6086 7549 7658 11 11  75% Mod 2479 2378 23 752 4 35  50% Mod 667 774 24 896 22 18 25% Mod 480 541 57 1557 43 115  10% Mod 838 956 159 2755 144 123  1%Mod 1108 1197 235 3415 88 270  0.1% Mod 1338 1177 191 2873 37 363  0%Mod 1463 1666 215 3793 74 429 Control 3272 3603 16 1557 731 9066 G-CSFmCherry 0 0 2 645 0 0 Untreated N/A N/A 1 1 0 8

Example 65 Innate Immune Response Study in BJ Fibroblasts

A. Single Transfection

Human primary foreskin fibroblasts (BJ fibroblasts) were obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, catalog #30-2003) supplementedwith 10% fetal bovine serum at 37° C., under 5% CO₂. BJ fibroblasts wereseeded on a 24-well plate at a density of 300,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 21438; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1) fully modified with 5-methylcytosineand pseudouridine (Gen1) or fully modified with 5-methylcytosine andN1-methyl-pseudouridine (Gen2) having Cap0, Cap1 or no cap wastransfected using Lipofectamine 2000 (Invitrogen, catalog #11668-019),following manufacturer's protocol. Control samples of poly I:C(PIC),Lipofectamine 2000 (Lipo), natural luciferase mRNA (mRNA sequence shownin SEQ ID NO: 21446; polyA tail of approximately 160 nucleotides notshown in sequence; 5′ cap, Cap1) and natural G-CSF mRNA were alsotransfected. The cells were harvested after 18 hours, the total RNA wasisolated and DNASE® treated using the RNeasy micro kit (catalog #74004)following the manufacturer's protocol. 100 ng of total RNA was used forcDNA synthesis using High Capacity cDNA Reverse Transcription kit(catalog #4368814) following the manufacturer's protocol. The cDNA wasthen analyzed for the expression of innate immune response genes byquantitative real time PCR using SybrGreen in a Biorad CFX 384instrument following manufacturer's protocol. Table 99 shows theexpression level of innate immune response transcripts relative tohouse-keeping gene HPRT (hypoxanthine phosphoribosytransferase) and isexpressed as fold-induction relative to HPRT. In the table, the panel ofstandard metrics includes: RIG-I is retinoic acid inducible gene 1, IL6is interleukin-6, OAS-1 is oligoadenylate synthetase 1, IFNb isinterferon-beta, AIM2 is absent in melanoma-2, IFIT-1 isinterferon-induced protein with tetratricopeptide repeats 1, PKR isprotein kinase R, TNFa is tumor necrosis factor alpha and IFNa isinterferon alpha.

TABLE 99 Innate Immune Response Transcript Levels Form. RIG-I IL6 OAS-1IFNb AIM2 IFIT-1 PKR TNFa IFNa Natural 71.5 20.6 20.778 11.440 0.251151.218 16.001 0.526 0.067 Luciferase Natural 73.3 47.1 19.359 13.6150.264 142.011 11.667 1.185 0.153 G-CSF PIC 30.0 2.8 8.628 1.523 0.10071.914 10.326 0.264 0.063 G-CSF 0.81 0.22 0.080 0.009 0.008 2.220 1.5920.090 0.027 Gen1-UC G-CSF 0.54 0.26 0.042 0.005 0.008 1.314 1.568 0.0880.038 Gen1- Cap0 G-CSF 0.58 0.30 0.035 0.007 0.006 1.510 1.371 0.0900.040 Gen1- Cap1 G-CSF 0.21 0.20 0.002 0.007 0.007 0.603 0.969 0.1290.005 Gen2-UC G-CSF 0.23 0.21 0.002 0.0014 0.007 0.648 1.547 0.121 0.035Gen2- Cap0 G-CSF 0.27 0.26 0.011 0.004 0.005 0.678 1.557 0.099 0.037Gen2- Cap1 Lipo 0.27 0.53 0.001 0 0.007 0.954 1.536 0.158 0.064

B. Repeat Transfection

Human primary foreskin fibroblasts (BJ fibroblasts) were obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, catalog #30-2003) supplementedwith 10% fetal bovine serum at 37° C., under 5% CO₂. BJ fibroblasts wereseeded on a 24-well plate at a density of 300,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 21438; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1) unmodified, fully modified with5-methylcytosine and pseudouridine (Gen1) or fully modified with5-methylcytosine and N1-methyl-pseudouridine (Gen2) was transfecteddaily for 5 days following manufacturer's protocol. Control samples ofLipofectamine 2000 (L2000) and mCherry mRNA (mRNA sequence shown in SEQID NO: 21439; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytidine andpseudouridine) were also transfected daily for 5 days. The results areshown in Table 100.

Unmodified mRNA showed a cytokine response in interferon-beta (IFN-beta)and interleukin-6 (IL-6) after one day. mRNA modified with at leastpseudouridine showed a cytokine response after 2-3 days whereas mRNAmodified with 5-methylcytosine and N1-methyl-pseudouridine showed areduced response after 3-5 days.

TABLE 100 Cytokine Response Formulation Transfection IFN-beta (pg/ml)IL-6 (pg/ml) G-CSF unmodified 6 hours 0 3596 Day 1 1363 15207 Day 2 23812415 Day 3 225 5017 Day 4 363 4267 Day 5 225 3094 G-CSF Gen 1 6 hours 03396 Day 1 38 3870 Day 2 1125 16341 Day 3 100 25983 Day 4 75 18922 Day 5213 15928 G-CSF Gen 2 6 hours 0 3337 Day 1 0 3733 Day 2 150 974 Day 3213 4972 Day 4 1400 4122 Day 5 350 2906 mCherry 6 hours 0 3278 Day 1 2383893 Day 2 113 1833 Day 3 413 25539 Day 4 413 29233 Day 5 213 20178L2000 6 hours 0 3270 Day 1 13 3933 Day 2 388 567 Day 3 338 1517 Day 4475 1594 Day 5 263 1561

Example 66 In vivo detection of Innate Immune Response

In an effort to distringuish the importance of different chemicalmodification of mRNA on in vivo protein production and cytokine responsein vivo, female BALB/C mice (n=5) are injected intramuscularly withG-CSF mRNA (G-CSF mRNA unmod) (mRNA sequence shown in SEQ ID NO: 21438;polyA tail of approximately 160 nucleotides not shown in sequence;) witha 5′ cap of Cap1, G-CSF mRNA fully modified with 5-methylcytosine andpseudouridine (G-CSF mRNA 5mc/pU), G-CSF mRNA fully modified with5-methylcytosine and N1-methyl-pseudouridine with (G-CSF mRNA 5mc/N1pU)or without a 5′ cap (G-CSF mRNA 5mc/N1 pU no cap) or a control of eitherR848 or 5% sucrose as described in Table 101.

TABLE 101 Dosing Chart Formulation Route Dose (ug/mouse) Dose (ul) G-CSFmRNA I.M. 200 50 unmod G-CSF mRNA I.M. 200 50 5mc/pU G-CSF mRNA I.M. 20050 5mc/N1pU G-CSF mRNA I.M. 200 50 5mc/N1pU no cap R848 I.M. 75 50 5%sucrose I.M. — 50 Untreated I.M. — —

Blood is collected at 8 hours after dosing. Using ELISA the proteinlevels of G-CSF, TNF-alpha and IFN-alpha is determined by ELISA. 8 hoursafter dosing, muscle is collected from the injection site andquantitative real time polymerase chain reaction (QPCR) is used todetermine the mRNA levels of RIG-I, PKR, AIM-2, IFIT-1, OAS-2, MDA-5,IFN-beta, TNF-alpha, IL-6, G-CSF, CD45 in the muscle.

Example 67 In vivo detection of Innate Immune Response Study

Female BALB/C mice (n=5) were injected intramuscularly with G-CSF mRNA(G-CSF mRNA unmod) (mRNA sequence shown in SEQ ID NO: 21438; polyA tailof approximately 160 nucleotides not shown in sequence;) with a 5′ capof Cap 1, G-CSF mRNA fully modified with 5-methylcytosine andpseudouridine (G-CSF mRNA 5mc/pU), G-CSF mRNA fully modified with5-methylcytosine and N1-methyl-pseudouridine with (G-CSF mRNA 5mc/N1pU)or without a 5′ cap (G-CSF mRNA 5mc/N1 pU no cap) or a control of eitherR848 or 5% sucrose as described in Table 102. Blood is collected at 8hours after dosing and using ELISA the protein levels of G-CSF andinterferon-alpha (IFN-alpha) is determined by ELISA and are shown inTable 102.

As shown in Table 102, unmodified, 5mc/pU, and 5mc/N1pU modified G-CSFmRNA resulted in human G-CSF expression in mouse serum. The uncapped5mC/N1pU modified G-CSF mRNA showed no human G-CSF expression in serum,highlighting the importance of having a 5′ cap structure for proteintranslation.

As expected, no human G-CSF protein was expressed in the R848, 5%sucrose only, and untreated groups. Importantly, significant differenceswere seen in cytokine production as measured by mouse IFN-alpha in theserum. As expected, unmodified G-CSF mRNA demonstrated a robust cytokineresponse in vivo (greater than the R848 positive control). The 5mc/pUmodified G-CSF mRNA did show a low but detectable cytokine response invivo, while the 5mc/N1pU modified mRNA showed no detectable IFN-alpha inthe serum (and same as vehicle or untreated animals).

Also, the response of 5mc/N1pU modified mRNA was the same regardless ofwhether it was capped or not. These in vivo results reinforce theconclusion that 1) that unmodified mRNA produce a robust innate immuneresponse, 2) that this is reduced, but not abolished, through 100%incorporation of 5mc/pU modification, and 3) that incorporation of5mc/N1pU modifications results in no detectable cytokine response.

Lastly, given that these injections are in 5% sucrose (which has noeffect by itself), these result should accurately reflect theimmunostimulatory potential of these modifications.

From the data it is evident that N1pU modified molecules produce moreprotein while concomitantly having little or no effect on IFN-alphaexpression. It is also evident that capping is required for proteinproduction for this chemical modification.

The Protein Cytokine Ratio of 748 as compared to the PC Ratio for theunmodified mRNA (PC=9) means that this chemical modification is farsuperior as related to the effects or biological implications associatedwith IFN-alpha.

TABLE 102 Human G-CSF and Mouse IFN-alpha in serum G-CSF IFN-alpha DoseDose protein expression PC Formulation Route (ug/mouse) (ul) (pg/ml)(pg/ml) Ratio G-CSF mRNA unmod I.M. 200 50 605.6 67.01 9 G-CSF mRNA 5mc/pU I.M. 200 50 356.5 8.87 40 G-CSF mRNA 5 mc/N1pU I.M. 200 50 748.1 0748 G-CSF mRNA 5 mc/N1pU I.M. 200 50 6.5 0 6.5 no cap R848 I.M. 75 503.4 40.97 .08 5% sucrose I.M. — 50 0 1.49 0 Untreated I.M. — — 0 0 0

Example 68 In Vivo Delivery of Modified RNA

Protein production of modified mRNA was evaluated by delivering modifiedG-CSF mRNA or modified Factor IX mRNA to female Sprague Dawley rats(n=6). Rats were injected with 400 ug in 100 ul of G-CSF mRNA (mRNAsequence shown in SEQ ID NO: 21438; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (G-CSF Gen1), G-CSF mRNA fullymodified with 5-methylcytosine and N1-methyl-pseudouridine (G-CSF Gen2)or Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 1622; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (Factor IX Gen1)reconstituted from the lyophilized form in 5% sucrose. Blood wascollected 8 hours after injection and the G-CSF protein level in serumwas measured by ELISA. Table 103 shows the G-CSF protein levels in serumafter 8 hours.

These results demonstrate that both G-CSF Gen 1 and G-CSF Gen 2 modifiedmRNA can produce human G-CSF protein in a rat following a singleintramuscular injection, and that human G-CSF protein production isimproved when using Gen 2 chemistry over Gen 1 chemistry.

TABLE 103 G-CSF Protein in Rat Serum (I.M. Injection Route) FormulationG-CSF protein (pg/ml) G-CSF Gen1 19.37 G-CSF Gen2 64.72 Factor IX Gen 12.25

Example 69 Chemical Modification: In Vitro Studies

A. In vitro Screening in PBMC

500 ng of G-CSF (mRNA sequence shown in SEQ ID NO: 21438; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) mRNAfully modified with the chemical modification outlined Tables 104 and105 was transfected with 0.4 uL Lipofectamine 2000 into peripheral bloodmononuclear cells (PBMC) from three normal blood donors. Control samplesof LPS, R848, P(I)P(C) and mCherry (mRNA sequence shown in SEQ ID NO:21439; polyA tail of approximately 160 nucleotides not shown insequence, 5′ cap, Capl; fully modified with 5-methylcytosine andpseudouridine) were also analyzed. The supernatant was harvested andstored frozen until analyzed by ELISA to determine the G-CSF proteinexpression, and the induction of the cytokines interferon-alpha (IFN-a)and tumor necrosis factor alpha (TNF-a). The protein expression of G-CSFis shown in Table 104, the expression of IFN-α and TNF-a is shown inTable 105.

The data in Table 104 demonstrates that many, but not all, chemicalmodifications can be used to productively produce human G-CSF in PBMC.Of note, 100% N¹-methyl-pseudouridine substitution demonstrates thehighest level of human G-CSF production (almost 10-fold higher thanpseudouridine itself). When N¹-methyl-pseudouridine is used incombination with 5-methylcytidine a high level of human G-CSF protein isalso produced (this is also higher than when pseudouridine is used incombination with 5 methylcytidine).

Given the inverse relationship between protein production and cytokineproduction in PBMC, a similar trend is also seen in Table 105, where100% substitution with Nl-methyl-pseudouridine results no cytokineinduction (similar to transfection only controls) and pseudouridineshows detectable cytokine induction which is above background.

Other modifications such as N⁶-methyladenosine and alpha-thiocytidineappear to increase cytokine stimulation.

TABLE 104 Chemical Modifications and G-CSF Protein Expression G-CSFProtein Expression (pg/ml) Donor Donor Donor Chemical Modifications 1 23 Pseudouridine 2477 1,909 1,498 5-methyluridine 318 359 345N1-methyl-pseudouridine 21,495 16,550 12,441 2-thiouridine 932 1,000 6004-thiouridine 5 391 218 5-methoxyuridine 2,964 1,832 1,8005-methylcytosine and pseudouridine (1^(st) set) 2,632 1,955 1,3735-methylcytosine and N1-methyl- 10,232 7,245 6,214 pseudouridine (1^(st)set) 2′Fluoroguanosine 59 186 177 2′Fluorouridine 118 209 1915-methylcytosine and pseudouridine (2^(nd) set) 1,682 1,382 1,0365-methylcytosine and N1-methyl- 9,564 8,509 7,141 pseudouridine (2^(nd)set) 5-bromouridine 314 482 291 5-(2-carbomethoxyvinyl)uridine 77 286177 5-[3(1-E-propenylamino)uridine 541 491 550 α-thiocytidine 105 264245 5-methylcytosine and pseudouridine (3^(rd) set) 1,595 1,432 955N1-methyladenosine 182 177 191 N6-methyladenosine 100 168 2005-methylcytidine 291 277 359 N4-acetylcytidine 50 136 365-formylcytidine 18 205 23 5-methylcytosine and pseudouridine (4^(th)set) 264 350 182 5-methylcytosine and N1-methyl- 9,505 6,927 5,405pseudouridine (4^(th) set) LPS 1,209 786 636 mCherry 5 168 164 R848 709732 636 P(I)P(C) 5 186 182

TABLE 105 Chemical Modifications and Cytokine Expression IFN-α TNF-αExpression (pg/ml) Expression (pg/ml) Chemical Donor Donor Donor DonorDonor Donor Modifications 1 2 3 1 2 3 Pseudouridine 120 77 171 36 81 1265-methyluridine 245 135 334 94 100 157 N1-methyl- 26 75 138 101 106 134pseudouridine 2-thiouridine 100 108 154 133 133 141 4-thiouridine 463258 659 169 126 254 5-methoxyuridine 0 64 133 39 74 111 5-methylcytosine88 94 148 64 89 121 and pseudouridine (1^(st) set) 5-methylcytosine 0 60136 54 79 126 and N1-methyl- pseudouridine (1^(st) set) 2′Fluoroguanosine 107 97 194 91 94 141 2′ Fluorouridine 158 103 178 164121 156 5-methylcytosine 133 92 167 99 111 150 and pseudouridine (2^(nd)set) 5-methylcytosine 0 66 140 54 97 149 and N1-methyl- pseudouridine(2^(nd) set) 5-bromouridine 95 86 181 87 106 157 5-(2- 0 61 130 40 81116 carbomethoxyvinyl) uridine 5-[3(1-E- 0 58 132 71 90 119propenylamino) uridine α-thiocytidine 1,138 565 695 300 273 2775-methylcytosine 88 75 150 84 89 130 and pseudouridine (3^(rd) set) N1-methyladenosine 322 255 377 256 157 294 N6-methyladenosine 1,935 1,0651,492 1,080 630 857 5-methylcytidine 643 359 529 176 136 193N4-acetylcytidine 789 593 431 263 67 207 5-formylcytidine 180 93 88 13630 40 5-methylcytosine 131 28 18 53 24 29 and pseudouridine (4^(th) set)5-methylcytosine 0 0 0 36 14 13 and N1-methyl- pseudouridine (4^(th)set) LPS 0 67 146 7,004 3,974 4,020 mCherry 100 75 143 67 100 133 R848674 619 562 11,179 8,546 9,907 P(I)P(C) 470 117 362 249 177 197

B. In vitro Screening in HeLa Cells

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, VA) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand

Island, N.Y.) and seeded in a total volume of 100u1EMEM medium(supplemented with 10% FCS and 1× Glutamax) per well in a 96-well cellculture plate (Corning, Manassas, Va.). The cells were grown at 37oG in5% CO₂ atmosphere overnight. Next day, 83 ng of Luciferase modified RNA(mRNA sequence shown in SEQ ID NO: 21446; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap1) with the chemicalmodification described in Table 106, were diluted in 10 ul final volumeof OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).

Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 0.2 ul were diluted in 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature.

After 18 to 22 hours of incubation cells expressing luciferase werelysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The lysate volumes were adjusted ordiluted until no more than 2 mio relative light units (RLU) per wellwere detected for the strongest signal producing samples and the RLUsfor each chemistry tested are shown in Table 106. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.).The background signal of theplates without reagent was about 200 relative light units per well.

These results demonstrate that many, but not all, chemical modificationscan be used to productively produce human G-CSF in HeLa cells. Of note,100% N1-methyl-pseudouridine substitution demonstrates the highest levelof human G-CSF production.

TABLE 106 Relative Light Units of Luciferase Chemical Modification RLUN6-methyladenosine (m6a) 534 5-methylcytidine (m5c) 138,428N4-acetylcytidine (ac4c) 235,412 5-formylcytidine (f5c) 4365-methylcytosine/pseudouridine, test A1 48,6595-methylcytosine/N1-methyl-pseudouridine, test A1 190,924 Pseudouridine655,632 1-methylpseudouridine (m1u) 1,517,998 2-thiouridine (s2u) 33875-methoxyuridine (mo5u) 253,719 5-methylcytosine/pseudouridine, test B1317,744 5-methylcytosine/N1-methyl-pseudouridine, test B1 265,8715-Bromo-uridine 43,276 5 (2 carbovinyl) uridine 531 5 (3-1E propenylAmino) uridine 446 5-methylcytosine/pseudouridine, test A2 295,8245-methylcytosine/N1-methyl-pseudouridine, test A2 233,9215-methyluridine 50,932 α-Thio-cytidine 26,3585-methylcytosine/pseudouridine, test B2 481,4775-methylcytosine/N1-methyl-pseudouridine, test B2 271,9895-methylcytosine/pseudouridine, test A3 438,8315-methylcytosine/N1-methyl-pseudouridine, test A3 277,499 UnmodifiedLuciferase 234,802

C. In vitro Screening in Rabbit Reticulocyte Lysates

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) wasmodified with the chemical modification listed in Table 107 and werediluted in sterile nuclease-free water to a final amount of 250 ng in 10ul. The diluted luciferase was added to 40 ul of freshly prepared RabbitReticulocyte Lysate and the in vitro translation reaction was done in astandard 1.5 mL polypropylene reaction tube (Thermo Fisher Scientific,Waltham, Mass.) at 30° C. in a dry heating block. The translation assaywas done with the Rabbit Reticulocyte Lysate (nuclease-treated) kit(Promega, Madison, Wis.) according to the manufacturer's instructions.The reaction buffer was supplemented with a one-to-one blend of providedamino acid stock solutions devoid of either Leucine or Methionineresulting in a reaction mix containing sufficient amounts of both aminoacids to allow effective in vitro translation.

After 60 minutes of incubation, the reaction was stopped by placing thereaction tubes on ice. Aliquots of the in vitro translation reactioncontaining luciferase modified RNA were transferred to white opaquepolystyrene 96-well plates (Corning, Manassas, Va.) and combined with100 ul complete luciferase assay solution (Promega, Madison, Wis.). Thevolumes of the in vitro translation reactions were adjusted or diluteduntil no more than 2 mio relative light units (RLUs) per well weredetected for the strongest signal producing samples and the RLUs foreach chemistry tested are shown in Table 107. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.).The background signal of theplates without reagent was about 200 relative light units per well.

These cell-free translation results very nicely correlate with theprotein production results in HeLa, with the same modificationsgenerally working or not working in both systems. One notable exceptionis 5-formylcytidine modified luciferase mRNA which worked in thecell-free translation system, but not in the HeLa cell-basedtransfection system. A similar difference between the two assays wasalso seen with 5-formylcytidine modified G-CSF mRNA.

TABLE 107 Relative Light Units of Luciferase Chemical Modification RLUN6-methyladenosine (m6a) 398 5-methylcytidine (m5c) 152,989N4-acetylcytidine (ac4c) 60,879 5-formylcytidine (f5c) 55,2085-methylcytosine/pseudouridine, test A1 349,3985-methylcytosine/N1-methyl-pseudouridine, test A1 205,465 Pseudouridine587,795 1-methylpseudouridine (m1u) 589,758 2-thiouridine (s2u) 7085-methoxyuridine (mo5u) 288,647 5-methylcytosine/pseudouridine, test B1454,662 5-methylcytosine/N1-methyl-pseudouridine, test B1 223,7325-Bromo-uridine 221,879 5 (2 carbovinyl) uridine 225 5 (3-1E propenylAmino) uridine 211 5-methylcytosine/pseudouridine, test A2 558,7795-methylcytosine/N1-methyl-pseudouridine, test A2 333,0825-methyluridine 214,680 α-Thio-cytidine 123,8785-methylcytosine/pseudouridine, test B2 487,8055-methylcytosine/N1-methyl-pseudouridine, test B2 154,0965-methylcytosine/pseudouridine, test A3 413,5355-methylcytosine/N1-methyl-pseudouridine, test A3 292,954 UnmodifiedLuciferase 225,986

Example 70 Chemical Modification: In vivo Studies

A. In vivo Screening of G-CSF Modified mRNA

Balb-C mice (n=4) are intramuscularly injected in each leg with modifiedG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 21438; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),fully modified with the chemical modifications outlined in Table 108, isformulated in 1×PBS. A control of luciferase modified mRNA (mRNAsequence shown in SEQ ID NO: 21446; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified withpseudouridine and 5-methylcytosine) and a control of PBS are alsotested. After 8 hours serum is collected to determine G-CSF proteinlevels cytokine levels by ELISA.

TABLE 108 G-CSF mRNA Chemical Modifications G-CSF Pseudouridine G-CSF5-methyluridine G-CSF 2-thiouridine G-CSF 4-thiouridine G-CSF5-methoxyuridine G-CSF 2′-fluorouridine G-CSF 5-bromouridine G-CSF5-[3(1-E-propenylamino)uridine) G-CSF alpha-thio-cytidine G-CSF5-methylcytidine G-CSF N4-acetylcytidine G-CSF Pseudouridine and5-methylcytosine G-CSF N1-methyl-pseudouridine and 5-methylcytosineLuciferase Pseudouridine and 5-methylcytosine PBS None

B. In vivo Screening of Luciferase Modified mRNA

Balb-C mice (n=4) were subcutaneously injected with 200 ul containing 42to 103 ug of modified luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1), fully modified with the chemical modificationsoutlined in Table 109, was formulated in 1×PBS. A control of PBS wasalso tested. The dosages of the modified luciferase mRNA is alsooutlined in Table 109. 8 hours after dosing the mice were imaged todetermine luciferase expression. Twenty minutes prior to imaging, micewere injected intraperitoneally with a D-luciferin solution at 150mg/kg. Animals were then anesthetized and images were acquired with anIVIS Lumina II imaging system (Perkin Elmer). Bioluminescence wasmeasured as total flux (photons/second) of the entire mouse.

As demonstrated in Table 109, all luciferase mRNA modified chemistriesdemonstrated in vivo activity, with the exception of 2′-fluorouridine.In addition 1-methylpseudouridine modified mRNA demonstrated very highexpression of luciferase (5-fold greater expression than pseudouridinecontaining mRNA).

TABLE 109 Luciferase Screening Dose Dose Luciferase Chemical (ug) ofvolume expression mRNA Modifications mRNA (ml) (photon/second)Luciferase 5-methylcytidine 83 0.72 1.94E+07 LuciferaseN4-acetylcytidine 76 0.72 1.11E07  Luciferase Pseudouridine 95 1.201.36E+07 Luciferase 1-methylpseudouridine 103  0.72 7.40E+07 Luciferase5-methoxyuridine 95 1.22 3.32+07   Luciferase 5-methyluridine 94 0.867.42E+06 Luciferase 5-bromouridine 89 1.49 3.75E+07 Luciferase2′-fluoroguanosine 42 0.72 5.88E+05 Luciferase 2′-fluorocytidine 47 0.724.21E+05 Luciferase 2′-flurorouridine 59 0.72 3.47E+05 PBS None — 0.723.16E+05

Example 71 In vivo Screening of Combination Luciferase Modified mRNA

Balb-C mice (n=4) were subcutaneously injected with 200 ul of 100 ug ofmodified luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1), fully modified with the chemical modifications outlined in Table110, was formulated in 1× PBS. A control of PBS was also tested. 8 hoursafter dosing the mice were imaged to determine luciferase expression.Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse.

As demonstrated in Table 110, all luciferase mRNA modified chemistries(in combination) demonstrated in vivo activity. In addition the presenceof N1-methyl-pseudouridine in the modified mRNA (with N4-acetylcytidineor 5 methylcytidine) demonstrated higher expression than when the samecombinations where tested using with pseudouridine. Taken together,these data demonstrate that N1-methyl-pseudouridine containingluciferase mRNA results in improved protein expression in vivo whetherused alone (Table 109) or when used in combination with other modifiednulceotides (Table 110).

TABLE 110 Luciferase Screening Combinations Luciferase expression(photon/ mRNA Chemical Modifications second) LuciferaseN4-acetylcytidine/pseudouridine 4.18E+06 LuciferaseN4-acetylcytidine/N1-methyl-pseudouridine 2.88E+07 Luciferase5-methylcytidine/5-methoxyuridine 3.48E+07 Luciferase5-methylcytidine/5-methyluridine 1.44E+07 Luciferase5-methylcytidine/where 50% of the uridine 2.39E+06 is replaced with2-thiouridine Luciferase 5-methylcytidine/pseudouridine 2.36E+07Luciferase 5-methylcytidine/N1-methyl-pseudouridine 4.15E+07 PBS None3.59E+05

Example 72 Innate Immune Response in BJ Fibroblasts

Human primary foreskin fibroblasts (BJ fibroblasts) are obtained fromAmerican Type Culture Collection (ATCC) (catalog #CRL-2522) and grown inEagle's Minimum Essential Medium (ATCC, cat#30-2003) supplemented with10% fetal bovine serum at 37° C., under 5% CO2. BJ fibroblasts areseeded on a 24-well plate at a density of 130,000 cells per well in 0.5ml of culture medium. 250 ng of modified G-CSF mRNA (mRNA sequence shownin SEQ ID NO: 21438; polyA tail of approximately 160 nucleotides notshown in sequence; 5′ cap, Cap1) fully modified with 5-methylcytosineand pseudouridine (Gen1) or fully modified with 5-methylcytosine andN1-methyl-pseudouridine (Gen2) is transfected using Lipofectamine 2000(Invitrogen, cat#11668-019), following manufacturer's protocol. Controlsamples of Lipofectamine 2000 and unmodified G-CSF mRNA (natural G-CSF)are also transfected. The cells are transfected for five consecutivedays. The transfection complexes are removed four hours after each roundof transfection.

The culture supernatant is assayed for secreted G-CSF (R&D Systems,catalog #DCS50), tumor necrosis factor-alpha (TNF-alpha) and interferonalpha (IFN-alpha by ELISA every day after transfection followingmanufacturer's protocols. The cells are analyzed for viability usingCELL TITER GLO® (Promega, catalog #G7570) 6 hrs and 18 hrs after thefirst round of transfection and every alternate day following that. Atthe same time from the harvested cells, total RNA is isolated andtreated with DNASE® using the RNAEASY micro kit (catalog #74004)following the manufacturer's protocol. 100 ng of total RNA is used forcDNA synthesis using the High Capacity cDNA Reverse Transcription kit(Applied Biosystems, cat #4368814) following the manufacturer'sprotocol. The cDNA is then analyzed for the expression of innate immuneresponse genes by quantitative real time PCR using SybrGreen in a BioradCFX 384 instrument following the manufacturer's protocol.

Example 73 In Vitro Transcription with Wild-Type T7 Polymerase

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 21438; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) werefully modified with different chemistries and chemistry combinationslisted in Tables 111-114 using wild-type T7 polymerase as previouslydescribed.

The yield of the translation reactions was determined byspectrophometric measurement (OD260) and the yield for Luciferase isshown in Table 111 and G-CSF is shown in Table 113.

The luciferase and G-CSF modified mRNA were also subjected to anenzymatic capping reaction and each modified mRNA capping reaction wasevaluated for yield by spectrophometic measurement (OD260) and correctsize assessed using bioanalyzer. The yield from the capping reaction forluciferase is shown in Table 112 and G-CSF is shown in Table 114.

TABLE 111 In vitro transcription chemistry for Luciferase ChemicalModification Yield (mg) N6-methyladenosine 0.99 5-methylcytidine 1.29N4-acetylcytidine 1.0 5-formylcytidine 0.55 Pseudouridine 2.0N1-methyl-pseudouridine 1.43 2-thiouridine 1.56 5-methoxyuridine 2.355-methyluridine 1.01 α-Thio-cytidine 0.83 5-Br-uridine (5Bru) 1.96 5 (2carbomethoxyvinyl) uridine 0.89 5 (3-1E propenyl Amino) uridine 2.01N4-acetylcytidine/pseudouridine 1.34N4-acetylcytidine/N1-methyl-pseudouridine 1.265-methylcytidine/5-methoxyuridine 1.38 5-methylcytidine/5-bromouridine0.12 5-methylcytidine/5-methyluridine 2.97 5-methylcytidine/half of theuridines 1.59 are modified with 2-thiouridine5-methylcytidine/2-thiouridine 0.90 5-methylcytidine/pseudouridine 1.835-methylcytidine/N1-methyl-pseudouridine 1.33

TABLE 112 Capping chemistry and yield for Luciferase modified mRNAChemical Modification Yield (mg) 5-methylcytidine 1.02 N4-acetylcytidine0.93 5-formylcytidine 0.55 Pseudouridine 2.07 N1-methyl-pseudouridine1.27 2-thiouridine 1.44 5-methoxyuridine 2 5-methyluridine 0.8α-Thio-cytidine 0.74 5-Br-uridine (5Bru) 1.29 5 (2 carbomethoxyvinyl)uridine 0.54 5 (3-1E propenyl Amino) uridine 1.39N4-acetylcytidine/pseudouridine 0.99N4-acetylcytidine/N1-methyl-pseudouridine 1.085-methylcytidine/5-methoxyuridine 1.13 5-methylcytidine/5-methyluridine1.08 5-methylcytidine/half of the uridines 1.2 are modified with2-thiouridine 5-methylcytidine/2-thiouridine 1.275-methylcytidine/pseudouridine 1.195-methylcytidine/N1-methyl-pseudouridine 1.04

TABLE 113 In vitro transcription chemistry and yield for G-CSF modifiedmRNA Chemical Modification Yield (mg) N6-methyladenosine 1.575-methylcytidine 2.05 N4-acetylcytidine 3.13 5-formylcytidine 1.41Pseudouridine 4.1 N1-methyl-pseudouridine 3.24 2-thiouridine 3.465-methoxyuridine 2.57 5-methyluridine 4.27 4-thiouridine 1.452′-F-uridine 0.96 α-Thio-cytidine 2.29 2′-F-guanosine 0.6N-1-methyladenosine 0.63 5-Br-uridine (5Bru) 1.08 5 (2carbomethoxyvinyl) uridine 1.8 5 (3-1E propenyl Amino) uridine 2.09N4-acetylcytidine/pseudouridine 1.72N4-acetylcytidine/N1-methyl-pseudouridine 1.375-methylcytidine/5-methoxyuridine 1.85 5-methylcytidine/5-methyluridine1.56 5-methylcytidine/half of the uridines 1.84 are modified with2-thiouridine 5-methylcytidine/2-thiouridine 2.535-methylcytidine/pseudouridine 0.63 N4-acetylcytidine/2-thiouridine 1.3N4-acetylcytidine/5-bromouridine 1.375-methylcytidine/N1-methyl-pseudouridine 1.25N4-acetylcytidine/pseudouridine 2.24

TABLE 114 Capping chemistry and yield for G-CSF modified mRNA ChemicalModification Yield (mg) N6-methyladenosine 1.04 5-methylcytidine 1.08N4-acetylcytidine 2.73 5-formylcytidine 0.95 Pseudouridine 3.88N1-methyl-pseudouridine 2.58 2-thiouridine 2.57 5-methoxyuridine 2.055-methyluridine 3.56 4-thiouridine 0.91 2′-F-uridine 0.54α-Thio-cytidine 1.79 2′-F-guanosine 0.14 5-Br-uridine (5Bru) 0.79 5 (2carbomethoxyvinyl) uridine 1.28 5 (3-1E propenyl Amino) uridine 1.78N4-acetylcytidine/pseudouridine 0.29N4-acetylcytidine/N1-methyl-pseudouridine 0.335-methylcytidine/5-methoxyuridine 0.91 5-methylcytidine/5-methyluridine0.61 5-methylcytidine/half of the uridines 1.24 are modified with2-thiouridine 5-methylcytidine/pseudouridine 1.08N4-acetylcytidine/2-thiouridine 1.34 N4-acetylcytidine/5-bromouridine1.22 5-methylcytidine/N1-methyl-pseudouridine 1.56

Example 74 In Vitro Transcription with Mutant T7 Polymerase

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 21438; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) werefully modified with different chemistries and chemistry combinationslisted in Tables 115-118 using a mutant T7 polymerase (Durascribe® T7Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.).

The yield of the translation reactions was determined byspectrophometric measurement (OD260) and the yield for Luciferase isshown in Tables 115 and G-CSF is shown in Tables 117.

The luciferase and G-CSF modified mRNA were also subjected to anenzymatic capping reaction and each modified mRNA capping reaction wasevaluated for yield by spectrophometic measurement (OD260) and correctsize assessed using bioanalyzer. The yield from the capping reaction forluciferase is shown in Table 116 and G-CSF is shown in Table 118.

TABLE 115 In vitro transcription chemistry and yield for Luciferasemodified mRNA Chemical Modification Yield (ug) 2′Fluorocytosine 71.42′Fluorouridine 57.5 5-methylcytosine/pseudouridine, test A 26.45-methylcytosine/N1-methyl-pseudouridine, test A 73.3N1-acetylcytidine/2-fluorouridine 202.2 5-methylcytidine/2-fluorouridine131.9 2-fluorocytosine/pseudouridine 119.32-fluorocytosine/N1-methyl-pseudouridine 107.02-fluorocytosine/2-thiouridine 34.7 2-fluorocytosine/5-bromouridine 81.02-fluorocytosine/2-fluorouridine 80.4 2-fluoroguanine/5-methylcytosine61.2 2-fluoroguanine/5-methylcytosine/pseudouridine 65.02-fluoroguanine/5-methylcytidine/N1-methyl-pseudouridine 41.22-fluoroguanine/pseudouridine 79.12-fluoroguanine/N1-methyl-pseudouridine 74.65-methylcytidine/pseudouridine, test B 91.85-methylcytidine/N1-methyl-pseudouridine, test B 72.4 2′fluoroadenosine190.98

TABLE 116 Capping chemistry and yield for Luciferase modified mRNAChemical Modification Yield (ug) 2′Fluorocytosine 19.2 2′Fluorouridine16.7 5-methylcytosine/pseudouridine, test A 7.05-methylcytosine/N1-methyl-pseudouridine, test A 21.5N1-acetylcytidine/2-fluorouridine 47.5 5-methylcytidine/2- fluorouridine53.2 2-fluorocytosine/pseudouridine 58.42-fluorocytosine/N1-methyl-pseudouridine 26.22-fluorocytosine/2-thiouridine 12.9 2-fluorocytosine/5-bromouridine 26.52-fluorocytosine/2-fluorouridine 35.7 2-fluoroguanine/5-methylcytosine24.7 2-fluoroguanine/5-methylcytosine/pseudouridine 32.32-fluoroguanine/5-methylcytidine/N1-methyl-pseudouridine 31.32-fluoroguanine/pseudouridine 20.92-fluoroguanine/N1-methyl-pseudouridine 29.85-methylcytidine/pseudouridine, test B 58.25-methylcytidine/N1-methyl-pseudouridine, test B 44.4

TABLE 117 In vitro transcription chemistry and yield for G-CSF modifiedmRNA Chemical Modification Yield (ug) 2′Fluorocytosine 56.52′Fluorouridine 79.4 5-methylcytosine/pseudouridine, test A 21.25-methylcytosine/N1-methyl-pseudouridine, test A 77.1N1-acetylcytidine/2-fluorouridine 168.6 5-methylcytidine/2-fluorouridine134.7 2-fluorocytosine/pseudouridine 97.82-fluorocytosine/N1-methyl-pseudouridine 103.12-fluorocytosine/2-thiouridine 58.8 2-fluorocytosine/5-bromouridine 88.82-fluorocytosine/2-fluorouridine 93.9 2-fluoroguanine/5-methylcytosine97.3 2-fluoroguanine/5-methylcytosine/pseudouridine 96.02-fluoroguanine/5-methylcytidine/N1-methyl-pseudouridine 82.02-fluoroguanine/pseudouridine 68.02-fluoroguanine/N1-methyl-pseudouridine 59.35-methylcytidine/pseudouridine, test B 58.75-methylcytidine/N1-methyl-pseudouridine, test B 78.0

TABLE 118 Capping chemistry and yield for G-CSF modified mRNA ChemicalModification Yield (ug) 2′Fluorocytosine 16.9 2′Fluorouridine 17.05-methylcytosine/pseudouridine, test A 10.65-methylcytosine/N1-methyl-pseudouridine, test A 22.7N1-acetylcytidine/2-fluorouridine 19.9 5-methylcytidine/2-fluorouridine21.3 2-fluorocytosine/pseudouridine 65.22-fluorocytosine/N1-methyl-pseudouridine 58.92-fluorocytosine/2-thiouridine 41.2 2-fluorocytosine/5-bromouridine 35.82-fluorocytosine/2-fluorouridine 36.7 2-fluoroguanine/5-methylcytosine36.6 2-fluoroguanine/5-methylcytosine/pseudouridine 37.32-fluoroguanine/5-methylcytidine/N1-methyl-pseudouridine 30.72-fluoroguanine/pseudouridine 29.02-fluoroguanine/N1-methyl-pseudouridine 22.75-methylcytidine/pseudouridine, test B 60.45-methylcytidine/N1-methyl-pseudouridine, test B 33.0

Example 75 2′O-methyl and 2′Fluoro Compounds

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) wereproduced as fully modified versions with the chemistries in Table 119and transcribed using mutant T7 polymerase (Durascribe® T7 Transcriptionkit (Cat. No. DS010925) (Epicentre®, Madison, Wis.). 2′fluoro-containing mRNA were made using Durascribe T7, however,2′Omethyl-containing mRNA could not be transcribed using Durascribe T7.

Incorporation of 2′Omethyl modified mRNA might possibly be accomplishedusing other mutant T7 polymerases (Nat. Biotechnol. (2004) 22:1155-1160;Nucleic Acids Res. (2002) 30:e138) or U.S. Pat. No. 7,309,570, thecontents of each of which are incorporated herein by reference in theirentirety. Alternatively, 2′OMe modifications could be introducedpost-transcriptionally using enzymatic means.

Introduction of modifications on the 2′ group of the sugar has manypotential advantages. 2′OMe substitutions, like 2′ fluoro substitutionsare known to protect against nucleases and also have been shown toabolish innate immune recognition when incorporated into other nucleicacids such as siRNA and anti-sense (incorporated in its entirety,Crooke, ed. Antisense Drug Technology, 2^(nd) edition; Boca Raton: CRCpress).

The 2′Fluoro-modified mRNA were then transfected into HeLa cells toassess protein production in a cell context and the same mRNA were alsoassessed in a cell-free rabbit reticulocyte system. A control ofunmodified luciferase (natural luciferase) was used for bothtranscription experiments, a control of untreated and mock transfected(Lipofectamine 2000 alone) were also analyzed for the HeLa transfectionand a control of no RNA was analyzed for the rabbit reticulysates.

For the HeLa transfection experiments, the day before transfection,20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested bytreatment with Trypsin-EDTA solution (LifeTechnologies, Grand Island,N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplementedwith 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate(Corning, Manassas, Va.). The cells were grown at 37oG in 5% CO₂atmosphere overnight. Next day, 83 ng of the 2′ fluoro-containingluciferase modified RNA (mRNA sequence shown in SEQ ID NO: 21446; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) with the chemical modification described in Table 119, werediluted in 1 0 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul were diluted in 10 ul finalvolume of OPTI-MEM. After 5 minutes of incubation at room temperature,both solutions were combined and incubated an additional 15 minute atroom temperature. Then the 20 ul combined solution was added to the 100ul cell culture medium containing the HeLa cells and incubated at roomtemperature. After 18 to 22 hours of incubation cells expressingluciferase were lysed with 100 ul of Passive Lysis Buffer (Promega,Madison, Wis.) according to manufacturer instructions. Aliquots of thelysates were transferred to white opaque polystyrene 96-well plates(Corning, Manassas, Va.) and combined with 100 ul complete luciferaseassay solution (Promega, Madison, Wis.). The lysate volumes wereadjusted or diluted until no more than 2 mio relative light units (RLU)per well were detected for the strongest signal producing samples andthe RLUs for each chemistry tested are shown in Table 119. The platereader was a BioTek Synergy H1 (BioTek, Winooski, Vt.).The backgroundsignal of the plates without reagent was about 200 relative light unitsper well.

For the rabbit reticulocyte lysate assay, 2′-fluoro-containingluciferase mRNA were diluted in sterile nuclease-free water to a finalamount of 250 ng in 10 ul and added to 40 ul of freshly prepared RabbitReticulocyte Lysate and the in vitro translation reaction was done in astandard 1.5 mL polypropylene reaction tube (Thermo Fisher Scientific,Waltham, Mass.) at 30° C. in a dry heating block. The translation assaywas done with the Rabbit Reticulocyte Lysate (nuclease-treated) kit(Promega, Madison, Wis.) according to the manufacturer's instructions.The reaction buffer was supplemented with a one-to-one blend of providedamino acid stock solutions devoid of either Leucine or Methionineresulting in a reaction mix containing sufficient amounts of both aminoacids to allow effective in vitro translation. After 60 minutes ofincubation, the reaction was stopped by placing the reaction tubes onice.

Aliquots of the in vitro translation reaction containing luciferasemodified RNA were transferred to white opaque polystyrene 96-well plates(Corning, Manassas, Va.) and combined with 100 ul complete luciferaseassay solution (Promega, Madison, Wis.). The volumes of the in vitrotranslation reactions were adjusted or diluted until no more than 2 miorelative light units (RLUs) per well were detected for the strongestsignal producing samples and the RLUs for each chemistry tested areshown in Table 120. The plate reader was a BioTek Synergy H1 (BioTek,Winooski, Vt.). The background signal of the plates without reagent wasabout 160 relative light units per well.

As can be seen in Table 119 and 120, multiple 2′Fluoro-containingcompounds are active in vitro and produce luciferase protein.

TABLE 119 HeLa Cells Concentration Volume Yield Chemical Modification(ug/ml) (ul) (ug) RLU 2′Fluoroadenosine 381.96 500 190.98 388.52′Fluorocytosine 654.56 500 327.28 2420 2′Fluoroguanine 541.795 500270.90 11,705.5 2′Flurorouridine 944.005 500 472.00 6767.5 Naturalluciferase N/A N/A N/A 133,853.5 Mock N/A N/A N/A 340 Untreated N/A N/AN/A 238

TABLE 120 Rabbit Reticulysates Chemical Modification RLU2′Fluoroadenosine 162 2′Fluorocytosine 208 2′Fluoroguanine 371,5092′Flurorouridine 258 Natural luciferase 2,159,968 No RNA 156

Example 76 Luciferase in HeLa Cells Using a Combination of Modifications

To evaluate using of 2′ fluoro-modified mRNA in combination with othermodification a series of mRNA were transcribed using either wild-type T7polymerase (non-fluoro-containing compounds) or using mutant T7polymerases (fluyoro-containing compounds) as described in Example 75.All modified mRNA were tested by in vitro transfection in HeLa cells.

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37oG in 5% CO₂ atmosphere overnight. Next day, 83 ng ofLuciferase modified RNA (mRNA sequence shown in SEQ ID NO: 21446; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) with the chemical modification described in Table 121, werediluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul were diluted in 10 ul finalvolume of OPTI-MEM. After 5 minutes of incubation at room temperature,both solutions were combined and incubated an additional 15 minute atroom temperature. Then the 20 ul combined solution was added to the 100ul cell culture medium containing the HeLa cells and incubated at roomtemperature.

After 18 to 22 hours of incubation cells expressing luciferase werelysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The lysate volumes were adjusted ordiluted until no more than 2 mio relative light units (RLU) per wellwere detected for the strongest signal producing samples and the RLUsfor each chemistry tested are shown in Table 121. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.).The background signal of theplates without reagent was about 200 relative light units per well.

As evidenced in Table 121, most combinations of modifications resultedin mRNA which produced functional luciferase protein, including all thenon-fluoro containing compounds and many of the combinations containing2′ fluoro modifications.

TABLE 121 Luciferase Chemical Modification RLUN4-acetylcytidine/pseudouridine 113,796N4-acetylcytidine/N1-methyl-pseudouridine 316,3265-methylcytidine/5-methoxyuridine 24,9485-methylcytidine/5-methyluridine 43,675 5-methylcytidine/half of theuridines modified with 50% 41,601 2-thiouridine5-methylcytidine/2-thiouridine 1,102 5-methylcytidine/pseudouridine51,035 5-methylcytidine/N1-methyl-pseudouridine 152,151N4-acetylcytidine/2′Fluorouridine triphosphate 2885-methylcytidine/2′Fluorouridine triphosphate 269 2′Fluorocytosinetriphosphate/pseudouridine 260 2′Fluorocytosinetriphosphate/N1-methyl-pseudouridine 412 2′Fluorocytosinetriphosphate/2-thiouridine 427 2′Fluorocytosinetriphosphate/5-bromouridine 253 2′Fluorocytosinetriphosphate/2′Fluorouridine triphosphate 184 2′Fluoroguaninetriphosphate/5-methylcytidine 321 2′Fluoroguaninetriphosphate/5-methylcytidine/Pseudouridine 2072′Fluoroguanine/5-methylcytidine/N1-methyl-psuedouridine 2352′Fluoroguanine/pseudouridine 2182′Fluoroguanine/N1-methyl-psuedouridine 2475-methylcytidine/pseudouridine, test A 13,8335-methylcytidine/N1-methyl-pseudouridine, test A 598 2′Fluorocytosinetriphosphate 201 2′Fluorouridine triphosphate 3055-methylcytidine/pseudouridine, test B 115,4015-methylcytidine/N1-methyl-pseudouridine, test B 21,034 Naturalluciferase 30,801 Untreated 344 Mock 262

Example 77 G-CSF In Vitro Transcription

To assess the activity of all our different chemical modifications inthe context of a second open reading frame, we replicated experimentspreviously conducted using luciferase mRNA, with human G-CSF mRNA. G-CSFmRNA (mRNA sequence shown in SEQ ID NO: 21438; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) werefully modified with the chemistries in Tables 122 and 123 usingwild-type T7 polymerase (for all non-fluoro-containing compounds) ormutant T7 polymerase (for all fluoro-containing compounds). The mutantT7 polymerase was obtained commercially (Durascribe® T7 Transcriptionkit (Cat. No. DS010925) (Epicentre®, Madison, Wis.).

The modified RNA in Tables 122 and 123 were transfected in vitro in HeLacells or added to rabbit reticulysates (250 ng of modified mRNA) asindicated. A control of untreated, mock transfected (transfectionreagent alone), G-CSF fully modified with 5-methylcytosine andN1-methyl-pseudouridine or luciferase control (mRNA sequence shown inSEQ ID NO: 21446; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine were also analyzed. The expression of G-CSFprotein was determined by ELISA and the values are shown in Tables 122and 123. In Table 122, “NT” means not tested.

As shown in Table 123, many, but not all, chemical modificationsresulted in human G-CSF protein production. These results fromcell-based and cell-free translation systems correlate very nicely withthe same modifications generally working or not working in both systems.One notable exception is 5-formylcytidine modified G-CSF mRNA whichworked in the cell-free translation system, but not in the HeLacell-based transfection system. A similar difference between the twoassays was also seen with 5-formylcytidine modified luciferase mRNA.

As demonstrated in Table 123, many, but not all, G-CSF mRNA modifiedchemistries (when used in combination) demonstrated in vivo activity. Inaddition the presence of N1-methyl-pseudouridine in the modified mRNA(with N4-acetylcytidine or methylcytidine) demonstrated higherexpression than when the same combinations where tested using withpseudouridine. Taken together, these data demonstrate thatN1-methyl-pseudouridine containing G-CSF mRNA results in improvedprotein expression in vitro.

TABLE 122 G-CSF Expression G-CSF protein G-CSF protein (pg/ml) Rabbit(pg/ml) reticulysates Chemical Modification HeLa cells cellsPseudouridine 1,150,909 147,875 5-methyluridine 347,045 147,2502-thiouridine 417,273 18,375 N1-methyl-pseudouridine NT 230,0004-thiouridine 107,273 52,375 5-methoxyuridine 1,715,909 201,7505-methylcytosine/pseudouridine, 609,545 119,750 Test A5-methylcytosine/N1-methyl- 1,534,318 110,500 pseudouridine, Test A2′-Fluoro-guanosine 11,818 0 2′-Fluoro-uridine 60,455 05-methylcytosine/pseudouridine, 358,182 57,875 Test B5-methylcytosine/N1-methyl- 1,568,636 76,750 pseudouridine, Test B5-Bromo-uridine 186,591 72,000 5-(2carbomethoxyvinyl) uridine 1,364 05-[3(1-E-propenylamino) uridine 27,955 32,625 α-thio-cytidine 120,45542,625 5-methylcytosine/pseudouridine, 882,500 49,250 Test CN1-methyl-adenosine 4,773 0 N6-methyl-adenosine 1,591 05-methyl-cytidine 646,591 79,375 N4-acetylcytidine 39,545 8,0005-formyl-cytidine 0 24,000 5-methylcytosine/pseudouridine, 87,045 47,750Test D 5-methylcytosine/N1-methyl- 1,168,864 97,125 pseudouridine, TestD Mock 909 682 Untreated 0 0 5-methylcytosine/N1-methyl- 1,106,591 NTpseudouridine, Control Luciferase control NT 0

TABLE 123 Combination Chemistries in HeLa cells G-CSF protein (pg/ml)Chemical Modification HeLa cells N4-acetylcytidine/pseudouridine 537,273N4-acetylcytidine/N1-methyl-pseudouridine 1,091,8185-methylcytidine/5-methoxyuridine 516,1365-methylcytidine/5-bromouridine 48,864 5-methylcytidine/5-methyluridine207,500 5-methylcytidine/2-thiouridine 33,409N4-acetylcytidine/5-bromouridine 211,591 N4-acetylcytidine/2-thiouridine46,136 5-methylcytosine/pseudouridine 301,3645-methylcytosine/N1-methyl-pseudouridine 1,017,727N4-acetylcytidine/2′Fluorouridine triphosphate 62,2735-methylcytidine/2′Fluorouridine triphosphate 49,318 2′Fluorocytosinetriphosphate/pseudouridine 7,955 2′Fluorocytosinetriphosphate/N1-methyl- 1,364 pseudouridine 2′Fluorocytosinetriphosphate/2-thiouridine 0 2′Fluorocytosinetriphosphate/5-bromouridine 1,818 2′Fluorocytosinetriphosphate/2′Fluorouridine 909 triphosphate 2′Fluoroguaninetriphosphate/5-methylcytidine 0 2′Fluoroguaninetriphosphate/5-methylcytidine/ 0 pseudouridine 2′Fluoroguaninetriphosphate/5-methylcytidine/ 1,818 N1 methylpseudouridine2′Fluoroguanine triphosphate/pseudouridine 1,136 2′Fluoroguaninetriphosphate/2′Fluorocytosine 0 triphosphate/N1-methyl-pseudouridine5-methylcytidine/pseudouridine 617,7275-methylcytidine/N1-methyl-pseudouridine 747,0455-methylcytidine/pseudouridine 475,4555-methylcytidine/N1-methyl-pseudouridine 689,0915-methylcytosine/N1-methyl-pseudouridine, 848,409 Control 15-methylcytosine/N1-methyl-pseudouridine, 581,818 Control 2 Mock 682Untreated 0 Luciferase 2′Fluorocytosine triphosphate 0 Luciferase2′Fluorouridine triphosphate 0

Example 78 Screening of Chemistries

The tables listed in below (Tables 124-126) summarize much of the invitro and in vitro screening data with the different compounds presentedin the previous examples. A good correlation exists between cell-basedand cell-free translation assays. The same chemistry substitutionsgenerally show good concordance whether tested in the context ofluciferase or G-CSF mRNA. Lastly, N1-methyl-pseudouridine containingmRNA show a very high level of protein expression with little to nodetectable cytokine stimulation in vitro and in vivo, and is superior tomRNA containing pseudouridine both in vitro and in vivo.

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 21438; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) weremodified with naturally and non-naturally occurring chemistriesdescribed in Tables 124 and 125 or combination chemistries described inTable 126 and tested using methods described herein.

In Tables 125 and 126, “*” refers to in vitro transcription reactionusing a mutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No.DS010925) (Epicentre®, Madison, Wis.); “**” refers to the second resultin vitro transcription reaction using a mutant T7 polymerase(Durascribe® T7 Transcription kit (Cat. No. DS010925) (Epicentre®,Madison, Wis.); “***” refers to production seen in cell freetranslations (rabbit reticulocyte lysates); the protein production ofHeLa is judged by “+,” “+/−” and “−”; when referring to G-CSF PBMC“++++” means greater than 6,000 pg/ml G-CSF, “+++” means greater than3,000 pg/ml G-CSF, “++” means greater than 1,500 pg/ml G-CSF, “+” meansgreater than 300 pg/ml G-CSF, “+/−” means 150-300 pg/ml G-CSF and thebackground was about 110 pg/ml; when referring to cytokine PBMC “++++”means greater than 1,000 pg/ml interferon-alpha (IFN-alpha), “+++” meansgreater than 600 pg/ml IFN-alpha, “++” means greater than 300 pg/mlIFN-alpha, “+” means greater than 100 pg/ml IFN-alpha, “−” means lessthan 100 pg/ml and the background was about 70 pg/ml; and “NT” means nottested. In Table 125, the protein production was evaluated using amutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No.DS010925) (Epicentre®, Madison, Wis.).

TABLE 124 Naturally Occurring Protein Protein Cytokines In In IVTProtein (G- (G- (G- Vivo Vivo Common Name IVT (G- (Luc; CSF; CSF; CSF;Protein Protein (symbol) (Luc) CSF) HeLa) HeLa) PBMC) PBMC) (Luc)(G-CSF) 1-methyladenosine Fail Pass NT − +/− ++ NT NT (m¹A) N6- PassPass − − +/− ++++ NT NT methyladenosine (m⁶A) 2′-O- Fail* Not NT NT NTNT NT NT methyladenosine Done (Am) 5-methylcytidine Pass Pass + + + ++ +NT (m⁵C) 2'-O- Fail* Not NT NT NT NT NT NT methylcytidine Done (Cm)2-thiocytidine Fail Fail NT NT NT NT NT NT (s²C) N⁴-acetylcytidine PassPass + + +/− +++ + NT (ac⁴C) 5-formylcytidine Pass Pass −*** −*** − + NTNT (f⁵C) 2′-O- Fail* Not NT NT NT NT NT NT methylguanosine Done (Gm)inosine (I) Fail Fail NT NT NT NT NT NT pseudouridine (Y) Pass Pass + +++ + + NT 5-methyluridine Pass Pass + + +/− + NT NT (m⁵U)2′-O-methyluridine Fail* Not NT NT NT NT NT NT (Um) Done 1- Pass Pass +Not ++++ − + NT methylpseudo- Done uridine (m¹Y) 2-thiouridine (s²U)Pass Pass − + + + NT NT 4-thiouridine (s⁴U) Fail Pass + +/− ++ NT NT5-methoxyuridine Pass Pass + + ++ − + NT (mo⁵U) 3-methyluridine FailFail NT NT NT NT NT NT (m³U)

TABLE 125 Non-Naturally Occurring Cytokines In Vivo IVT Protein ProteinProtein (G- In Vivo Protein Common IVT (G- (Luc; (G-CSF; (G-CSF; CSF;Protein (G- Name (Luc) CSF) HeLa) HeLa) PBMC) PBMC) (Luc) CSF) 2′-F-ara-Fail Fail NT NT NT NT NT NT guanosine 2′-F-ara- Fail Fail NT NT NT NT NTNT adenosine 2′-F-ara- Fail Fail NT NT NT NT NT NT cytidine 2′-F-ara-Fail Fail NT NT NT NT NT NT uridine 2′-F- Fail/ Pass/ +** +/− − + + NTguanosine Pass** Fail** 2′-F- Fail/ Fail/ −** NT NT NT NT NT adenosinePass** Fail** 2′-F-cytidine Fail/ Fail/ +** NT NT NT + NT Pass** Pass**2′-F-uridine Fail/ Pass/ +** + +/− + − NT Pass** Pass** 2′-OH-ara- FailFail NT NT NT NT NT NT guanosine 2′-OH-ara- Not Not NT NT NT NT NT NTadenosine Done Done 2′-OH-ara- Fail Fail NT NT NT NT NT NT cytidine2′-OH-ara- Fail Fail NT NT NT NT NT NT uridine 5-Br-Uridine PassPass + + + + + 5-(2- Pass Pass − − +/− − carbomethox yvinyl) Uridine5-[3-(1-E- Pass Pass − + + − Propenylamino) Uridine (aka Chem 5) N6-(19-Fail Fail NT NT NT NT NT NT Amino- pentaoxanona decyl) A 2- Fail Fail NTNT NT NT NT NT Dimethylamino guanosine 6-Aza- Fail Fail NT NT NT NT NTNT cytidine a-Thio- Pass Pass + + +/− +++ NT NT cytidine Pseudo- NT NTNT NT NT NT NT NT isocytidine 5-Iodo- NT NT NT NT NT NT NT NT uridinea-Thio- NT NT NT NT NT NT NT NT uridine 6-Aza-uridine NT NT NT NT NT NTNT NT Deoxy- NT NT NT NT NT NT NT NT thymidine a-Thio NT NT NT NT NT NTNT NT guanosine 8-Oxo- NT NT NT NT NT NT NT NT guanosine 06-Methyl- NTNT NT NT NT NT NT NT guanosine 7-Deaza- NT NT NT NT NT NT NT NTguanosine 6-Chloro- NT NT NT NT NT NT NT NT purine a-Thio- NT NT NT NTNT NT NT NT adenosine 7-Deaza- NT NT NT NT NT NT NT NT adenosine 5-iodo-NT NT NT NT NT NT NT NT cytidine

In Table 126, the protein production of HeLa is judged by “+,” “+/−” and“−”; when referring to G-CSF PBMC “++++” means greater than 6,000 pg/mlG-CSF, “+++” means greater than 3,000 pg/ml G-CSF, “++” means greaterthan 1,500 pg/ml G-CSF, “+” means greater than 300 pg/ml G-CSF, “+/−”means 150-300 pg/ml G-CSF and the background was about 110 pg/ml; whenreferring to cytokine PBMC “++++” means greater than 1,000 pg/mlinterferon-alpha (IFN-alpha), “+++” means greater than 600 pg/mlIFN-alpha, “++” means greater than 300 pg/ml IFN-alpha, “+” meansgreater than 100 pg/ml IFN-alpha, “−” means less than 100 pg/ml and thebackground was about 70 pg/ml; “WT” refers to the wild type T7polymerase, “MT” refers to mutant T7 polymerase (Durascribe® T7Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.) and“NT” means not tested.

TABLE 126 Combination Chemistry Protein Protein In IVT Protein (G- (G-Cytokines Vivo Cytidine Uridine IVT (G- (Luc; CSF; CSF; (G-CSF; Proteinanalog analog Purine Luc CSF) HeLa) HeLa) PBMC) PBMC) (Luc) N4- pseudo-A, G Pass Pass + + NT NT + acetylcytidine uridine WT WT N4- N1- A, GPass Pass + + NT NT + acetylcytidine methyl- WT WT pseudo- uridine 5- 5-A, G Pass Pass + + NT NT + methyl- methoxy- WT WT cytidine uridine 5- 5-A, G Pass Pass Not + NT NT methyl- bromo- WT WT Done cytidine uridine 5-5- A, G Pass Pass + + NT NT + methyl- methyl- WT WT cytidine uridine 5-50% 2- A, G Pass Pass + NT NT NT + methyl- thiouridine; WT WT cytidine50% uridine 5- 100% 2- A, G Pass Pass − + NT NT methyl- thiouridine WTWT cytidine 5- pseudo- A, G Pass Pass + + ++ + + methyl- uridine WT WTcytidine 5- N1- A, G Pass Pass + + ++++ − + methyl- methyl- WT WTcytidine pseudo- uridine N4- 2- A, G Not Pass Not + NT NT NT acetyl-thiouridine Done WT Done cytidine N4- 5- A, G Not Pass Not + NT NT NTacetyl- bromo- Done WT Done cytidine uridine N4- 2 A, G Pass Pass − + NTNT NT acetyl- Fluoro- cytidine uridine triphosphate 5- 2 A, G Pass Pass− + NT NT NT methyl- Fluoro- cytidine uridine triphosphate 2 pseudo- A,G Pass Pass − + NT NT NT Fluoro- uridine cytosine triphosphate 2 N1- A,G Pass Pass − +/− NT NT NT Fluoro- methyl- cytosine pseudo- triphosphateuridine 2 2- A, G Pass Pass − − NT NT NT Fluoro- thiouridine cytosinetriphosphate 2 5- A, G Pass Pass − +/− NT NT NT Fluoro- bromo- cytosineuridine triphosphate 2 2 A, G Pass Pass − +/− NT NT NT Fluoro- Fluoro-cytosine uridine triphosphate triphosphate 5- uridine A, 2 Pass Pass − −NT NT NT methyl- Fluoro cytidine GTP 5- pseudo- A, 2 Pass Pass − − NT NTNT methyl- uridine Fluoro cytidine GTP 5- NI- A, 2 Pass Pass − +/− NT NTNT methyl- methyl- Fluoro cytidine pseudo GTP uridine 2 pseudo- A, 2Pass Pass − +/− NT NT NT Fluoro- uridine Fluoro cytosine GTPtriphosphate 2 N1- A, 2 Pass Pass − − NT NT NT Fluoro- methyl- Fluorocytosine pseudo- GTP triphosphate uridine

Example 79. 2′ Fluoro Chemistries in PBMC

The ability of G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO:21438; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) to trigger innate an immune response wasdetermined by measuring interferon-alpha (IFN-alpha) and tumor necrosisfactor-alpha (TNF-alpha) production. Use of in vitro PBMC cultures is anaccepted way to measure the immunostimulatory potential ofoligonucleotides (Robbins et al., Oligonucleotides 2009 19:89-102) andtransfection methods are described herein. Shown in Table 127 are theaverage from 2 or 3 separate PBMC donors of the interferon-alpha(IFN-alpha) and tumor necrosis factor alpha (TNF-alpha) production overtime as measured by specific ELISA. Controls of R848, P(I)P(C), LPS andLipofectamine 2000 (L2000) were also analyzed.

With regards to innate immune recognition, while both modified mRNAchemistries largely prevented IFN-alpha and TNF-alpha productionrelative to positive controls (R848, P(I)P(C)), 2′ fluoro compoundsreduce IFN-alpha and TNF-alpha production even lower than othercombinations and N4-acetylcytidine combinations raised the cytokineprofile.

TABLE 127 IFN-alpha and TNF-alpha IFN-alpha: TNF-alpha: 3 Donor 2 DonorAverage Average (pg/ml) (pg/ml) L2000 1 361 P(I)P(C) 482 544 R848 458,235 LPS 0 6,889 N4-acetylcytidine/pseudouridine 694 528N4-acetylcytidine/N1-methyl-pseudouridine 307 2835-methylcytidine/5-methoxyuridine 0 411 5-methylcytidine/5-bromouridine0 270 5-methylcytidine/5-methyluridine 456 4285-methylcytidine/2-thiouridine 274 277 N4-acetylcytidine/2-thiouridine 0285 N4-acetylcytidine/5-bromouridine 44 4035-methylcytidine/pseudouridine 73 3325-methylcytidine/N1-methyl-pseudouridine 31 280N4-acetylcytidine/2′fluorouridine triphosphate 35 325-methylcytodine/2′fluorouridine triphosphate 24 0 2′fluorocytidinetriphosphate/N1-methyl- 0 11 pseudouridine 2′fluorocytidinetriphosphate/2-thiouridine 0 02′fluorocytidine/triphosphate5-bromouridine 12 2 2′fluorocytidinetriphosphate/2′fluorouridine 11 0 triphosphate 2′fluorocytidinetriphosphate/5-methylcytidine 14 23 2′fluorocytidine triphosphate/5- 621 methylcytidine/pseudouridine 2′fluorocytidine triphosphate/5- 3 15methylcytidine/N1-methyl-pseudouridine 2′fluorocytidinetriphosphate/pseudouridine 0 4 2′fluorocytidine triphosphate/N1-methyl-6 20 pseudouridine 5-methylcytidine/pseudouridine 82 185-methylcytidine/N1-methyl-pseudouridine 35 3

Example 80 Modified mRNA with a Tobacco Etch Virus 5′UTR

A 5′ untranslated region (UTR) may be provided as a flanking region.Multiple 5′ UTRs may be included in the flanking region and may be thesame or of different sequences. Any portion of the flanking regions,including none, may be codon optimized and any may independently containone or more different structural or chemical modifications, beforeand/or after codon optimization.

The 5′ UTR may comprise the 5′UTR from the tobacco etch virus (TEV).Variants of 5′ UTRs may be utilized wherein one or more nucleotides areadded or removed to the termini, including A, T, C or G.

Example 81 Expression of PLGA Formulated mRNA

A. Synthesis and Characterization of Luciferase PLGA Microspheres

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and N1-methyl-pseudouridine, modifiedwith 25% of uridine replaced with 2-thiouridine and 25% of cytosinereplaced with 5-methylcytosine, fully modified withN1-methyl-pseudouridine, or fully modified with pseudouridine wasreconstituted in 1× TE buffer and then formulated in PLGA microspheres.PLGA microspheres were synthesized using the water/oil/water doubleemulsification methods known in the art using PLGA-ester cap (Lactel,Cat# B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA),polyvinylalcohol (PVA) (Sigma, Cat#348406-25G, MW 13-23k)dichloromethane and water. Briefly, 0.4 ml of mRNA in TE buffer at 4mg/ml (W1) was added to 2 ml of PLGA dissolved in dichloromethane (DCM)(01) at a concentration of 200 mg/ml of PLGA. The W1/O1 emulsion washomogenized (IKA Ultra-Turrax Homogenizer, T18) for 30 seconds at speed5 (19,000 rpm). The W1/O1 emulsion was then added to 250 ml 1% PVA (W2)and homogenized for 1 minute at speed 5 (19,000 rpm). Formulations wereleft to stir for 3 hours, then passed through a 100 μm nylon meshstrainer (Fisherbrand Cell Strainer, Cat #22-363-549) to remove largeraggregates, and finally washed by centrifugation (10 min, 9,250 rpm, 4°C.). The supernatant was discarded and the PLGA pellets were resuspendedin 5-10 ml of water, which was repeated 2×. After washing andresuspension with water, 100-200 μl of a PLGA microspheres sample wasused to measure particle size of the formulations by laser diffraction(Malvern Mastersizer2000). The washed formulations were frozen in liquidnitrogen and then lyophilized for 2-3 days.

After lyophilization, ˜10 mg of PLGA MS were weighed out in 2 mleppendorf tubes and deformulated by adding 1 ml of DCM and letting thesamples shake for 2-6 hrs. The mRNA was extracted from the deformulatedPLGA micropsheres by adding 0.5 ml of water and shaking the sampleovernight. Unformulated luciferase mRNA in TE buffer (unformulatedcontrol) was spiked into DCM and went through the deformulation process(deformulation control) to be used as controls in the transfectionassay. The encapsulation efficiency, weight percent loading and particlesize are shown in Table 128. Encapsulation efficiency was calculated asmg of mRNA from deformulation of PLGA microspheres divided by theinitial amount of mRNA added to the formulation. Weight percent loadingin the formulation was calculated as mg of mRNA from deformulation ofPLGA microspheres divided by the initial amount of PLGA added to theformulation.

TABLE 128 PLGA Characteristics Theoretical Actual Encapsulation mRNAmRNA Particle Efficiency Loading Loading Size Chemical ModificationsSample ID (%) (wt %) (wt %) (D50, um) Fully modified with 5- 43-66A 45.80.4 0.18 33.4 methylcytosine and N1- 43-66B 29.6 0.12 27.7methyl-pseudouridine 43-66C 25.5 0.10 27.1 25% of uridine replaced43-67A 34.6 0.4 0.14 29.9 with 2-thiouridine and 43-67B 22.8 0.09 30.225% of cytosine replaced 43-67C 23.9 0.10 25.1 with 5-methylcytosineFully modified with N1- 43-69A 55.8 0.4 0.22 40.5 methyl-pseudouridine43-69B 31.2 0.12 41.1 43-69C 24.9 0.10 46.1 Fully modified with 43-68-149.3 0.4 0.20 34.8 pseudouridine 43-68-2 37.4 0.15 35.9 43-68-3 45.00.18 36.5

B. Protein Expression of modified mRNA Encapsulated in PLGA Microspheres

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100u1EMEM medium (supplemented with 10% FCS and 1× Glutamax) per well ina 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 83 ng ofthe deformulated luciferase mRNA PLGA microsphere samples, deformulatedluciferase mRNA control (Deform control), or unformulated luciferasemRNA control (Unfomul control) was diluted in a 10 ul final volume ofOPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as a transfectionreagent and 0.2u1 was diluted in a 10 ul final volume of OPTI-MEM. After5 min of incubation at room temperature, both solutions were combinedand incubated an additional 15 min at room temperature. Then 20 ul ofthe combined solution was added to 100 ul of cell culture mediumcontaining the HeLa cells. The plates were then incubated as describedbefore.

After an 18 to 22 hour incubation, cells expressing luciferase werelysed with 100u1 Passive Lysis Buffer (Promega, Madison, Wis.) accordingto manufacturer instructions. Aliquots of the lysates were transferredto white opaque polystyrene 96-well plates (Corning, Manassas, Va.) andcombined with 100u1 complete luciferase assay solution (Promega,Madison, Wis.). The background signal of the plates without reagent wasabout 200 relative light units per well. The plate reader was a BioTekSynergy H1 (BioTek, Winooski, Vt.).

Cells were harvested and the bioluminescence (in relative light units,RLU) for each sample is shown in Table 129. Transfection of thesesamples confirmed that the varied chemistries of luciferase mRNA isstill able to express luciferase protein after PLGA microsphereformulation.

TABLE 129 Chemical Modifications Chemical Biolum. Modifications SampleID (RLU) Fully modified with Deform contol 164266.5 5-methylcytosineUnformul control 113714 and N1-methyl- 43-66A 25174 pseudouridine 43-66B25359 43-66C 20060 25% of uridine Deform contol 90816.5 replaced with 2-Unformul control 129806 thiouridine and 25% 43-67A 38329.5 of cytosinereplaced 43-67B 8471.5 with 5- 43-67C 10991.5 methylcytosine Fullymodified with Deform contol 928093.5 N1-methyl- Unformul control1512273.5 pseudouridine 43-69A 1240299.5 43-69B 748667.5 43-69C 1193314Fully modified with Deform contol 154168 pseudouridine Unformul control151581 43-68-1 120974.5 43-68-2 107669 43-68-3 97226

Example 82 In vitro studies of Factor IX

A. Serum-Free Media

Human Factor IX mRNA (mRNA sequence shown in SEQ ID NO: 1622;

polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) wastransfected in serum-free media. The cell culture supernatant wascollected and subjected to trypsin digestion before undergoing2-dimensional HPLC separation of the peptides. Matrix-assisted laserdesorption/ionization was used to detect the peptides. 8 peptides weredetected and 7 of the detected peptides are unique to Factor IX. Theseresults indicate that the mRNA transfected in the serum-free media wasable to express full-length Factor IX protein.

B. Human Embryonic Kidney (HEK) 293A Cells

ng of codon optimized Human Factor IX mRNA (mRNA sequence shown in SEQID NO: 1622; fully modified with 5-methylcytosine and pseudouridine;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) was transfected into HEK 293A cells (150,000 cells/well)using Lipofectamine 2000 in DMEM in presence of 10% FBS. Thetransfection complexes were removed 3 hours after transfection. Cellswere harvested at 3, 6, 9, 12, 24, 48 and 72 hours after transfection.Total RNA was isolated and used for cDNA synthesis. The cDNA wassubjected to analysis by quantitative Real-Time PCR using codonoptimized Factor IXspecific primer set. Human hypoxanthinephosphoribosyltransfersase 1 (HPRT) level was used for normalization.The data is plotted as a percent of detectable mRNA considering the mRNAlevel as 100% at the 3 hour time point. The half-life of Factor IXmodified mRNA fully modified with 5-methylcytosine and pseudouridine inhuman embryonic kidney 293 (HEK293) cells is about 8-10 hours.

Example 83 Saline Formulation: Subcutaneous Administration

Human G-CSF modified mRNA (mRNA sequence shown in SEQ ID NO: 21438;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine) andhuman EPO modified mRNA (mRNA sequence shown in SEQ ID NO: 1638; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine), wereformulated in saline and delivered to mice via intramuscular (IM)injection at a dose of 100 ug.

Controls included Luciferase (mRNA sequence shown in SEQ ID NO: 21446;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine)) orthe formulation buffer (F.Buffer). The mice were bled at 13 hours afterthe injection to determine the concentration of the human polypeptide inserum in pg/mL. (G-CSF groups measured human G-CSF in mouse serum andEPO groups measured human EPO in mouse serum). The data are shown inTable 130.

mRNA degrades rapidly in serum in the absence of formulation suggestingthe best method to deliver mRNA to last longer in the system is byformulating the mRNA. As shown in Table 130, mRNA can be deliveredsubcutaneously using only a buffer formulation.

TABLE 130 Dosing Regimen Average Protein Dose Vol. Dosing Product pg/mL,Group Treatment (μl/mouse) Vehicle serum G-CSF G-CSF 100 F. buffer 45G-CSF Luciferase 100 F. buffer 0 G-CSF F. buffer 100 F. buffer 2.2 EPOEPO 100 F. buffer 72.03 EPO Luciferase 100 F. buffer 26.7 EPO F. buffer100 F. buffer 13.05

Example 84 Intravitreal Delivery

mCherry modified mRNA (mRNA sequence shown in SEQ ID NO: 21439; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) andluciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1; fully modified with 5-methylcytosine and pseudouridine) formulatedin saline was delivered intravitreally in rats as described in Table131. The sample was compared against a control of saline only deliveredintravitreally.

TABLE 131 Dosing Chart Dose Level Dose Treatment (μg modified volumeRight Eye Left Eye Group No. RNA/eye) (μL/eye) (OD) (OS) Control 0 5Delivery Delivery buffer only buffer only Modified RNA in 10 5 mCherryLuciferase delivery buffer

The formulation will be administered to the left or right eye of eachanimal on day 1 while the animal is anesthetized. On the day prior toadministration gentamicin ophthalmic ointment or solution was applied toboth eyes twice. The gentamicin ophthalmic ointment or solution was alsoapplied immediately following the injection and on the day following theinjection.Prior to dosing, mydriatic drops (1% tropicamide and/or 2.5%phenylephrine) are applied to each eye.

18 hours post dosing the eyes receiving the dose of mCherry and deliverybuffer are enucleated and each eye was separately placed in a tubecontaining 10 mL 4% paraformaldehyde at room temperature for overnighttissue fixation. The following day, eyes will be separately transferredto tubes containing 10 mL of 30% sucurose and stored at 21° C. untilthey were processed and sectioned. The slides prepared from differentsections were evaluated under F-microscopy. Postive expression was seenin the slides prepared with the eyes administered mCherry modified mRNAand the control showed no expression.

Example 85 In Vivo Cytokine Expression Study

Mice were injected intramuscularly with 200 ug of G-CSF modified mRNA(mRNA sequence shown in SEQ ID NO: 21438; polyA tail of approximately160 nucleotides not shown in sequence) which was unmodified with a 5′cap, Cap1 (unmodified), fully modified with 5-methylcytosine andpseudouridine and a 5′ cap, Cap1 (Gen1) or fully modified with5-methylcytosine and N1-methyl-pseudouridine and a 5′ cap, Cap1 (Gen2cap) or no cap (Gen2 uncapped). Controls of R-848, 5% sucrose anduntreated mice were also analyzed. After 8 hours serum was collectedfrom the mice and analyzed for interferon-alpha (IFN-alpha) expression.The results are shown in Table 132.

TABLE 132 IFN-alpha Expression Formulation IFN-alpha (pg/ml) G-CSFunmodified 67.012 G-CSF Gen1 8.867 G-CSF Gen2 cap 0 G-CSF Gen2 uncapped0 R-848 40.971 5% sucrose 1.493 Untreated 0

Example 86 In Vitro Expression of VEGF Modified mRNA

HEK293 cells were transfected with modified mRNA (mmRNA) VEGF-A (mRNAsequence shown in SEQ ID NO: 1672; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) which had been complexed withLipofectamine-2000 from Invitrogen (Carlsbad, Calif.) at theconcentration shown in Table 133. The protein expression was detected byELISA and the protein (pg/ml) is shown in Table 133 and FIG. 7.

TABLE 133 Protein Expression Amount 10 2.5 625 156 39 10 2 610Transfected ng ng pg pg pg pg pg fg Protein 10495 10038 2321.23 189.60 00 0 0 (pg/ml)

Example 87 In vitro Screening in HeLa Cells of GFP

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 37.5 ng or 75ng of Green Fluorescent protein (GFP) modified RNA (mRNA sequence shownin SEQ ID NO: 21448; polyA tail of approximately 160 nucleotides notshown in sequence; 5′ cap, Cap1) with the chemical modificationdescribed in Table 134, were diluted in 10 ul final volume of OPTI-MEM(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul were diluted in 10 ul final volume of OPTI-MEM. After 5minutes of incubation at room temperature, both solutions were combinedand incubated an additional 15 minute at room temperature. Then the 20ul combined solution was added to the 100 ul cell culture mediumcontaining the HeLa cells and incubated at room temperature.

After an 18 to 22 hour incubation cells expressing luciferase were lysedwith 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.) accordingto manufacturer instructions. Aliquots of the lysates were transferredto white opaque polystyrene 96-well plates (Corning, Manassas, Va.) andcombined with 100 ul complete luciferase assay solution (Promega,Madison, Wis.). The median fluorescence intensity (MFI) was determinedfor each chemistry and is shown in Table 134.

These results demonstrate that GFP fully modified withN1-methyl-pseudouridine and 5-methylcytosine produces more protein inHeLa cells compared to the other chemistry. Additionally the higher doseof GFP administered to the cells resulted in the highest MFI value.

TABLE 134 Mean Fluorescence Intensity 37.5 ng 75 ng Chemistry MFI MFI Nomodifications 97400 89500 5-methylcytosine/pseudouridine 324000 7150005-methylcytosine/N1-methyl-pseudouridine 643000 1990000

Example 88 Homogenization

Different luciferase mRNA solutions (as described in Table 135 where “X”refers to the solution containing that component) (mRNA sequence shownin SEQ ID NO: 21446; polyA tail of approximately 160 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytosineand pseudouridine) were evaluated to test the percent yield of thedifferent solutions, the integrity of the mRNA by bioanalyzer, and theprotein expression of the mRNA by in vitro transfection. The mRNAsolutions were prepared in water, 1× TE buffer at 4 mg/ml as indicatedin Table 135, and added to either dichloromethane (DCM) or DCMcontaining 200 mg/ml of poly(lactic-co-glycolic acid) (PLGA) (Lactel,Cat# B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA) to achieve afinal mRNA concentration of 0.8 mg/ml. The solutions requiringhomogenization were homogenized for 30 seconds at speed 5 (approximately19,000 rpm) (IKA Ultra-Turrax Homogenizer, T18). The mRNA samples inwater, dichloromethane and poly(lactic-co-glycolic acid) (PLGA) were notrecoverable (NR). All samples, except the NR samples, maintainedintegrity of the mRNA as determined by bioanalyzer (Bio-rad Experion).

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 250 ngof luciferase mRNA from the recoverable samples was diluted in a 10 ulfinal volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used as atransfection reagent and 0.2 ul was diluted in a 10 μl final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minutes at roomtemperature. Then 20 ul of the combined solution was added to 100 ul ofcell culture medium containing the HeLa cells. The plates were thenincubated as described before. Controls luciferase mRNA (luciferase mRNAformulated in saline) (Control) and untreated cells (Untreat.) were alsoevaluated. Cells were harvested and the bioluminescence average (inphotons/second) (biolum. (p/s)) for each signal is also shown in Table135. The recoverable samples all showed activity of luciferase mRNA whenanalyzed.

After an 18 to 22 hour incubation, cells expressing luciferase werelysed with 100 ul Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The background signal of the plateswithout reagent was about 200 relative light units per well. The platereader was a BioTek Synergy H1 (BioTek, Winooski, Vt.).

Cells were harvested and the bioluminescence average (in relative lightunits, RLU) (biolum. (RLU)) for each signal is also shown in Table 135.The recoverable samples all showed activity of luciferase mRNA whenanalyzed.

TABLE 135 Solutions Solution 1x TE DCM/ Homog- Yield Biolum. No. WaterBuffer DCM PLGA enizer (%) (RLU) 1 X 96 5423780 2 X X 95 4911950 3 X X92 2367230 4 X X 90 4349410 5 X X X 66 4145340 6 X X X 71 3834440 7 X XX NR n/a 8 X X X 24 3182080 9 X X NR n/a 10 X X 79 3276800 11 X X 795563550 12 X X 79 4919100 Control 2158060 Untreat. 3530

Example 89 TE Buffer and Water Evaluation

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) was reconstituted inwater or TE buffer as outlined in Table 136 and then formulated in PLGAmicrospheres. PLGA microspheres were synthesized using thewater/oil/water double emulsification methods known in the art usingPLGA (Lactel, Cat# B6010-2, inherent viscosity 0.55-0.75, 50:50 LA:GA),polyvinylalcohol (PVA) (Sigma, Cat#348406-25G, MW 13-23k)dichloromethane and water. Briefly, 0.2 to 0.6 ml of mRNA in water or TEbuffer at a concentration of 2 to 6 mg/ml (W1) was added to 2 ml of PLGAdissolved in dichloromethane (DCM) (01) at a concentration of 100 mg/mlof PLGA. The W1/O1 emulsion was homogenized (IKA Ultra-TurraxHomogenizer, T18) for 30 seconds at speed 5 (19,000 rpm). The W1/O1emulsion was then added to 250 ml 1% PVA (W2) and homogenized for 1minute at speed 5 (19,000 rpm).

Formulations were left to stir for 3 hours, then passed through a 100 μmnylon mesh strainer (Fisherbrand Cell Strainer, Cat #22-363-549) toremove larger aggregates, and finally washed by centrifugation (10 min,9,250 rpm, 4° C.). The supernatant was discarded and the PLGA pelletswere resuspended in 5-10 ml of water, which was repeated 2×. The washedformulations were frozen in liquid nitrogen and then lyophilized for 2-3days. After lyophilization, ˜10 mg of PLGA MS were weighed out in 2 mleppendorf tubes and deformulated by adding 1 ml of DCM and letting thesamples shake for 2-6 hrs. mRNA was extracted from the deformulated PLGAmicropsheres by adding 0.5 ml of water and shaking the sample overnight.Unformulated luciferase mRNA in water or TE buffer (deformulationcontrols) was spiked into DCM and went through the deformulation processto be used as controls in the transfection assay.

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO2 atmosphere overnight. The next day, 100 ngof the deformulated luciferase mRNA samples was diluted in a 10 ul finalvolume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine2000 (LifeTechnologies, Grand Island, N.Y.) was used as a transfectionreagent and 0.2 ul was diluted in a 10 ul final volume of OPTI-MEM.After 5 minutes of incubation at room temperature, both solutions werecombined and incubated an additional 15 minutes at room temperature.Then 20 ul of the combined solution was added to 100 ul of cell culturemedium containing the HeLa cells. The plates were then incubated asdescribed before.

After 18 to 22 hour incubation, cells expressing luciferase were lysedwith 100 ul Passive Lysis Buffer (Promega, Madison, Wis.) according tomanufacturer instructions. Aliquots of the lysates were transferred towhite opaque polystyrene 96-well plates (Corning, Manassas, Va.) andcombined with 100 ul complete luciferase assay solution (Promega,Madison, Wis.). The background signal of the plates without reagent wasabout 200 relative light units per well. The plate reader was a BioTekSynergy H1 (BioTek, Winooski, Vt.). To determine the activity of theluciferase mRNA from each formulation, the relative light units (RLU)for each formulation was divided by the RLU of the appropriate mRNAdeformulation control (mRNA in water or TE buffer). Table 136 shows theactivity of the luciferase mRNA. The activity of the luciferase mRNA inthe PLGA microsphere formulations (Form.) was substantitally improved byformulating in TE buffer versus water.

TABLE 136 Formulations W1 Theoretical Actual mRNA Solvent Total mRNAmRNA Activity conc. volume mRNA Loading Loading W1 (% of deform. Form.(mg/ml) (ul) (ug) (wt %) (wt %) Solvent control) PLGA A 4 400 1600 0.800.14 Water 12.5% PLGA B 4 200 800 0.40 0.13 Water 1.3% PLGA C 4 600 24001.20 0.13 Water 12.1% PLGA D 2 400 800 0.40 0.07 Water 1.3% PLGA E 6 4002400 1.20 0.18 TE Buffer 38.9% PLGA F 4 400 1600 0.80 0.16 TE Buffer39.7% PLGA G 4 400 1600 0.80 0.10 TE Buffer 26.6%

Example 90 Chemical Modifications on mRNA

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(Life Technologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 83 ng ofLuciferase modified RNA (mRNA sequence shown SEQ ID NO: 21446; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) with the chemical modification described in Table 137, werediluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul were diluted in 10 ul finalvolume of OPTI-MEM. After 5 minutes of incubation at room temperature,both solutions were combined and incubated an additional 15 minute atroom temperature. Then the 20 ul combined solution was added to the 100ul cell culture medium containing the HeLa cells and incubated at roomtemperature.

After 18 to 22 hours of incubation cells expressing luciferase werelysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The lysate volumes were adjusted ordiluted until no more than 2 mio relative light units (RLU) per wellwere detected for the strongest signal producing samples and the RLUsfor each chemistry tested are shown in Table 137. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.).The background signal of theplates without reagent was about 200 relative light units per well.

TABLE 137 Chemical Modifications Sample RLU Untreated 336 UnmodifiedLuciferase 33980 5-methylcytosine and pseudouridine 16012345-methylcytosine and N1-methyl-pseudouridine 421189 25% cytosinesreplaced with 5-methylcytosine 222114 and 25% of uridines replaced with2-thiouridine N1-methyl-pseudouridine 3068261 Pseudouridine 140234N4-Acetylcytidine 1073251 5-methoxyuridine 219657 5-Bromouridine 6787N4-Acetylcytidineand N1-methyl-pseudouridine 976219 5-methylcytosine and5-methoxyuridine 66621 5-methylcytosine and 2′fluorouridine 11333

Example 91 Intramuscular and Subcutaneous Administration of ModifiedmRNA

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) fully modified with 5-methylcytosine and pseudouridine (5mC/pU),fully modified with 5-methylcytosine and N1-methyl-pseudouridine(5mC/N1mpU), fully modified with pseudouridine (pU), fully modified withN1-methyl-pseudouridine (N1mpU) or modified where 25% of the cytosinesreplaced with 5-methylcytosine and 25% of the uridines replaced with2-thiouridine (5mC/s2U) formulated in PBS (pH 7.4) was administered toBalb-C mice intramuscularly or subcutaneously at a dose of 2.5 mg/kg.The mice were imaged at 2 hours, 8 hours, 24 hours, 48 hours, 72 hours,96 hours, 120 hours and 144 hours for intramuscular delivery and 2hours, 8 hours, 24 hours, 48 hours, 72 hours, 96 hours and 120 hours forsubcutaneous delivery. Twenty minutes prior to imaging, mice wereinjected intraperitoneally with a D-luciferin solution at 150 mg/kg.Animals were then anesthetized and images were acquired with an IVISLumina II imaging system (Perkin Elmer). Bioluminescence was measured astotal flux (photons/second) of the entire mouse. The average total flux(photons/second) for intramuscular administration is shown in Table 138and the average total flux (photons/second) for subcutaneousadministration is shown in Table 139. The background signal was 3.79E+05(p/s). The peak expression for intramuscular administration was seenbetween 24 and 48 hours for all chemistry and expression was stilldetected at 144 hours. For subcutaneous delivery the peak expression wasseen at 2-8 hours and expression was detected at 72 hours.

TABLE 138 Intramuscular Administration 5mC/ 5mC/ 5mC/ pU N1mpU s2U pUN1mpU Flux Flux Flux Flux Flux (p/s) (p/s) (p/s) (p/s) (p/s)  2 hours1.98E+07 4.65E+06 4.68E+06 2.33E+06 3.66E+07  8 hours 1.42E+07 3.64E+063.78E+06 8.07E+06 7.21E+07 24 hours 2.92E+07 1.22E+07 3.35E+07 1.01E+071.75E+08 48 hours 2.64E+07 1.01E+07 5.06E+07 7.46E+06 3.42E+08 72 hours2.18E+07 8.59E+06 3.42E+07 4.08E+06 5.83E+07 96 hours 2.75E+07 2.70E+062.38E+07 4.35E+06 7.15E+07 120 hours  2.19E+07 1.60E+06 1.54E+071.25E+06 3.87E+07 144 hours  9.17E+06 2.19E+06 1.14E+07 1.86E+065.04E+07

TABLE 139 Subcutaneous Administration 5mC/ 5mC/ 5mC/ pU N1mpU s2U pUN1mpU Flux Flux Flux Flux Flux (p/s) (p/s) (p/s) (p/s) (p/s)  2 hours5.26E+06 4.54E+06 9.34E+06 2.43E+06 2.80E+07  8 hours 2.32E+06 8.75E+058.15E+06 2.12E+06 3.09E+07 24 hours 2.67E+06 5.49E+06 3.80E+06 2.24E+061.48E+07 48 hours 1.22E+06 1.77E+06 3.07E+06 1.58E+06 1.24E+07 72 hours1.12E+06 8.00E+05 8.53E+05 4.80E+05 2.29E+06 96 hours 5.16E+05 5.33E+054.30E+05 4.30E+05 6.62E+05 120 hours  3.80E+05 4.09E+05 3.21E+056.82E+05 5.05E+05

Example 92 Osmotic Pump Study

Prior to implantation, an osmotic pump (ALZET® Osmotic Pump 2001D,DURECT Corp. Cupertino, Calif.) is loaded with the 0.2 ml of 1× PBS (pH7.4) (PBS loaded pump) or 0.2 ml of luciferase modified mRNA (mRNAsequence shown in SEQ ID NO: 21446; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and N1-methyl-pseudouridine) at 1 mg/ml in 1× PBS (pH7.4) (Luciferase loaded pump) and incubated overnight in 1× PBS (pH 7.4)at 37° C.

Balb-C mice (n=3) are implanted subcutaneously with either the PBSloaded pump or the luciferase loaded pump and imaged at 2 hours, 8 hoursand 24 hours. As a control a PBS loaded pump is implanted subcutaneouslyand the mice are injected subcutaneously with luciferase modified mRNAin 1× PBS (PBS loaded pump; SC Luciferase) or an osmotic pump is notimplanted and the mice are injected subcutaneously with luciferasemodified mRNA in 1× PBS (SC Luciferase). The luciferase formulations areoutlined in Table 140.

TABLE 140 Luciferase Formulations Conc Inj. Vol. Amt Dose Group Vehicle(mg/ml) (ul) (ug) (mg/kg) PBS loaded pump; PBS 1.00 50 50 2.5 SCLuciferase Luciferase loaded PBS 1.00 — 200 10.0 pump PBS loaded pumpPBS — — — — SC Luciferase PBS 1.00 50 50 2.5

Example 93 External Osmotic Pump Study

An external osmotic pump (ALZET® Osmotic Pump 2001D, DURECT Corp.Cupertino, Calif.) is loaded with the 0.2 ml of 1× PBS (pH 7.4) (PBSloaded pump) or 0.2 ml of luciferase modified mRNA (mRNA sequence shownin SEQ ID NO: 21446; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytosineand N1-methyl-pseudouridine) at 1 mg/ml in 1× PBS (pH 7.4) (luciferaseloaded pump) and incubated overnight in 1× PBS (pH 7.4) at 37° C.

Using a catheter connected to the external PBS loaded pump or theluciferase loaded pump Balb-C mice (n=3) are administered theformulation. The mice are imaged at 2 hours, 8 hours and 24 hours. As acontrol an external PBS loaded pump is used and the mice are injectedsubcutaneously with luciferase modified mRNA in 1× PBS (PBS loaded pump;SC Luciferase) or the external pump is not used and the mice are onlyinjected subcutaneously with luciferase modified mRNA in 1× PBS (SCLuciferase). Twenty minutes prior to imaging, mice are injectedintraperitoneally with a D-luciferin solution at 150 mg/kg. Animals arethen anesthetized and images are acquired with an IVIS Lumina II imagingsystem (Perkin Elmer). Bioluminescence is measured as total flux(photons/second) of the entire mouse. The luciferase formulations areoutlined in Table 141 and the average total flux (photons/second).

TABLE 141 Luciferase Formulations Conc Inj. Vol. Amt Dose Group Vehicle(mg/ml) (ul) (ug) (mg/kg) PBS loaded pump; PBS 1.00 50 50 2.5 SCLuciferase Luciferase loaded PBS 1.00 — 200 10.0 pump PBS loaded pumpPBS — — — — SC Luciferase PBS 1.00 50 50 2.5

Example 94 Fibrin Sealant Study

Fibrin sealant, such as Tisseel (Baxter Healthcare Corp., Deerfield,Ill.), is composed of fibrinogen and thrombin in a dual-barreledsyringe. Upon mixing, fibrinogen is converted to fibrin to form a fibrinclot in about 10 to 30 seconds. This clot can mimic the natural clottingmechanism of the body. Additionally a fibrin hydrogel is a threedimensional structure that can potentially be used in sustained releasedelivery. Currently, fibrin sealant is approved for application inhemostasis and sealing to replace conventional surgical techniques suchas suture, ligature and cautery.

The thrombin and fibrinogen components were loaded separately into adual barreled syringe. Balb-C mice (n=3) were injected subcutaneouslywith 50 ul of fibrinogen, 50 ul of thrombin and they were also injectedat the same site with modified luciferase mRNA (mRNA sequence shown inSEQ ID NO: 21446; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andN1-methyl-pseudouridine) (Tisseel+Luciferase), 50 ul of fibrinogen and50 ul thrombin (Tisseel) or modified luciferase mRNA (Luciferase). Theinjection of fibrinogen and thrombin was done simultaneously using thedual-barreled syringe. The SC injection of luciferase was done 15minutes after the fibrinogen/thrombin injection to allow the fibrinhydrogel to polymerize (Tisseel+Luciferase group). A control group ofuntreated mice were also evaluated. The mice were imaged at 5 hours and24 hours. Twenty minutes prior to imaging, mice were injectedintraperitoneally with a D-luciferin solution at 150 mg/kg. Animals werethen anesthetized and images were acquired with an IVIS Lumina IIimaging system (Perkin Elmer). Bioluminescence was measured as totalflux (photons/second) of the entire mouse. The luciferase formulationsare outlined in Table 142 and the average total flux (photons/second) isshown in Table 143. The fibrin sealant was found to not interfere withimaging and the injection of luciferase and Tisseel showed expression ofluciferase.

TABLE 142 Luciferase Formulations Conc Inj. Vol. Amt Dose Group Vehicle(mg/ml) (ul) (ug) (mg/kg) Tisseel + PBS 1.00 50 50 2.5 LuciferaseTisseel — — — — — Luciferase PBS 1.00 50 50 2.5 Untreated — — — — —

TABLE 143 Total Flux 5 Hours 24 Hours Group Flux (p/s) Flux (p/s)Tisseel + Luciferase 4.59E+05 3.39E+05 Tisseel 1.99E+06 1.06E+06Luciferase 9.94E+05 7.44E+05 Untreated 3.90E+05 3.79E+05

Example 95 Fibrin Containing mRNA Sealant Study

A. Modified mRNA and Calcium Chloride

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 21446; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridineis added to calcium chloride. The calcium chloride is then used toreconstitute thrombin. Fibrinogen is reconstituted with fibrinolysisinhibitor solution per the manufacturer's instructions. Thereconstituted thrombin containing modified mRNA and fibrinogen is loadedinto a dual barreled syringe. Mice are injected subcutaneously with 50ul of fibrinogen and 50 ul of thrombin containing modified mRNA or theywere injected with 50 ul of PBS containing an equivalent dose ofmodified luciferase mRNA. A control group of untreated mice is alsoevaluated. The mice are imaged at predetermined intervals to determinethe average total flux (photons/second).

B. Lipid Nanoparticle Formulated Modified mRNA and Calcium Chloride

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 21446; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridineis formulated in a lipid nanoparticle is added to calcium chloride. Thecalcium chloride is then used to reconstitute thrombin. Fibrinogen isreconstituted with fibrinolysis inhibitor solution per themanufacturer's instructions. The reconstituted thrombin containingmodified mRNA and fibrinogen is loaded into a dual barreled syringe.Mice are injected subcutaneously with 50 ul of fibrinogen and 50 ul ofthrombin containing modified mRNA or they were injected with 50 ul ofPBS containing an equivalent dose of modified luciferase mRNA. A controlgroup of untreated mice is also evaluated. The mice are imaged atpredetermined intervals to determine the average total flux(photons/second).

C. Modified mRNA and Fibrinogen

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 21446; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine or fully modified with Nl-methyl-pseudouridineis added to the fibrinolysis inhibitor solution. The fibrinolysisinhibitor solution is then used to reconstitute fibrinogen. Thrombin isreconstituted with the calcium chloride solution per the manufacturer'sinstructions. The reconstituted fibrinogen containing modified mRNA andthrombin is loaded into a dual barreled syringe. Mice are injectedsubcutaneously with 50 ul of thrombin and 50 ul of fibrinogen containingmodified mRNA or they were injected with 50 ul of PBS containing anequivalent dose of modified luciferase mRNA. A control group ofuntreated mice is also evaluated. The mice are imaged at predeterminedintervals to determine the average total flux (photons/second).

D. Lipid Nanoparticle Formualted Modified mRNA and Fibrinogen

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 21446; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridineis formulated in a lipid nanoparticle is added to the fibrinolysisinhibitor solution. The fibrinolysis inhibitor solution is then used toreconstitute fibrinogen. Thrombin is reconstituted with the calciumchloride solution per the manufacturer's instructions. The reconstitutedfibrinogen containing modified mRNA and thrombin is loaded into a dualbarreled syringe. Mice are injected subcutaneously with 50 ul ofthrombin and 50 ul of fibrinogen containing modified mRNA or they wereinjected with 50 ul of PBS containing an equivalent dose of modifiedluciferase mRNA. A control group of untreated mice is also evaluated.The mice are imaged at predetermined intervals to determine the averagetotal flux (photons/second).

E. Modified mRNA and Thrombin

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 21446; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridineis added to the reconstituted thrombin after it is reconstituted withthe calcium chloride per the manufactuer's instructions. Thefibrinolysis inhibitor solution is then used to reconstitute fibrinogenper the manufacturer's instructions. The reconstituted fibrinogen andthrombin containing modified mRNA is loaded into a dual barreledsyringe. Mice are injected subcutaneously with 50 ul of thrombincontaining modified mRNA and 50 ul of fibrinogen or they were injectedwith 50 ul of PBS containing an equivalent dose of modified luciferasemRNA. A control group of untreated mice is also evaluated. The mice areimaged at predetermined intervals to determine the average total flux(photons/second).

F. Lipid Nanoparticle Formualted Modified mRNA and Thrombin

Prior to reconstitution, luciferase mRNA (mRNA sequence shown in SEQ IDNO: 21446; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andN1-methyl-pseudouridine or fully modified with N1-methyl-pseudouridineis formulated in a lipid nanoparticle is added to the reconstitutedthrombin after it is reconstituted with the calcium chloride per themanufactuer's instructions. The fibrinolysis inhibitor solution is thenused to reconstitute fibrinogen per the manufacturer's instructions. Thereconstituted fibrinogen and thrombin containing modified mRNA is loadedinto a dual barreled syringe. Mice are injected subcutaneously with 50ul of thrombin containing modified mRNA and 50 ul of fibrinogen or theywere injected with 50 ul of PBS containing an equivalent dose ofmodified luciferase mRNA. A control group of untreated mice is alsoevaluated. The mice are imaged at predetermined intervals to determinethe average total flux (photons/second).

Example 96 Cationic Lipid Formulation of 5-Methylcytosine andN1-Methyl-Pseudouridine Modified mRNA

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and N1-methyl-pseudouridine wasformulated in the cationic lipids described in Table 144. Theformulations were administered intravenously (I.V.), intramuscularly(I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.

TABLE 144 Cationic Lipid Formulations Formulation NPA-126-1 NPA-127-1NPA-128-1 NPA-129-1 111612-B Lipid DLin-MC3- DLin-KC2- C12-200 DLinDMADODMA DMA DMA Lipid/mRNA 20:1 20:1 20:1 20:1 20:1 ratio (wt/wt) MeanSize 122 nm 114 nm 153 nm 137 nm 223.2 nm PDI: 0.13 PDI: 0.10 PDI: 0.17PDI: 0.09 PDI: 0.142 Zeta at pH 7.4 −1.4 mV −0.5 mV −1.4 mV 2.0 mV −3.09mV Encaps. 95% 77% 69% 80% 64% (RiboGr)

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 4.17E+05 p/s. The results of the imaging areshown in Table 145. In Table 145, “NT” means not tested.

TABLE 145 Flux DLin- DLin- MC3- KC2- Time DMA DMA C12-200 DLinDMA DODMARoute Point Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V. 2 hrs 1.92E+08 2.91E+08 1.08E+08 2.53E+07 8.40E+06 I.V.  8 hrs 1.47E+082.13E+08 3.72E+07 3.82E+07 5.62E+06 I.V. 24 hrs 1.32E+07 2.41E+075.35E+06 4.20E+06 8.97E+05 I.M.  2 hrs 8.29E+06 2.37E+07 1.80E+071.51E+06 NT I.M.  8 hrs 5.83E+07 2.12E+08 2.60E+07 1.99E+07 NT I.M. 24hrs 4.30E+06 2.64E+07 3.01E+06 9.46E+05 NT S.C.  2 hrs 1.90E+07 5.16E+078.91E+07 4.66E+06 9.61E+06 S.C.  8 hrs 7.74E+07 2.00E+08 4.58E+079.67E+07 1.90E+07 S.C. 24 hrs 7.49E+07 2.47E+07 6.96E+06 6.50E+061.28E+06

Example 97 Lipid Nanoparticle Intravenous Study

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) was formulated in alipid nanoparticle containing 50% DLin-MC3-DMA OR DLin-KC2-DMA asdescribed in Table 146, 38.5% cholesterol, 10% DSPC and 1.5% PEG. Theformulation was administered intravenously (I.V.) to Balb-C mice at adose of 0.5 mg/kg, 0.05 mg/kg, 0.005 mg/kg or 0.0005 mg/kg. Twentyminutes prior to imaging, mice were injected intraperitoneally with aD-luciferin solution at 150 mg/kg. Animals were then anesthetized andimages were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse.

TABLE 146 Formulations Formulation NPA-098-1 NPA-100-1 LipidDLin-KC2-DMA DLin-MC3-DMA Lipid/mRNA ratio (wt/wt) 20:1 20:1 Mean Size135 nm 152 nm PDI: 0.08 PDI: 0.08 Zeta at pH 7.4 −0.6 mV −1.2 mV Encaps.(RiboGr) 91% 94%

For DLin-KC2-DMA the mice were imaged at 2 hours, 8 hours, 24 hours, 72hours, 96 hours and 168 hours after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The background flux was about 3.66E+05 p/s.The results of the imaging are shown in Table 147. Organs were imaged at8 hours and the average total flux (photons/second) was measured for theliver, spleen, lung and kidney. A control for each organ was alsoanalyzed. The results are shown in Table 147. The peak signal for alldose levels was at 8 hours after administration. Also, distribution tothe various organs (liver, spleen, lung, and kidney) may be able to becontrolled by increasing or decreasing the LNP dose.

TABLE 147 Flux 0.5 mg/kg 0.05 mg/kg 0.005 mg/kg 0.0005 mg/kg Time PointFlux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) 2 hrs 3.54E+08 1.75E+072.30E+06 4.09E+05 8 hrs 1.67E+09 1.71E+08 9.81E+06 7.84E+05 24 hrs2.05E+08 2.67E+07 2.49E+06 5.51E+05 72 hrs 8.17E+07 1.43E+07 1.01E+063.75E+05 96 hrs 4.10E+07 9.15E+06 9.58E+05 4.29E+05 168 hrs 3.42E+079.15E+06 1.47E+06 5.29E+05

TABLE 148 Organ Flux Liver Spleen Lung Kidney Flux (p/s) Flux (p/s) Flux(p/s) Flux (p/s) 0.5 mg/kg 1.42E+08 4.86E+07 1.90E+05 3.20E+05 0.05mg/kg 7.45E+06 4.62E+05 6.86E+04 9.11E+04 0.005 mg/kg 3.32E+05 2.97E+041.42E+04 1.15E+04 0.0005 mg/kg 2.34E+04 1.08E+04 1.87E+04 9.78E+03Untreated 1.88E+04 1.02E+04 1.41E+04 9.20E+03

For DLin-MC3-DMA the mice were imaged at 2 hours, 8 hours, 24 hours, 48hours, 72 hours and 144 hours after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The background flux was about 4.51E+05 p/s.The results of the imaging are shown in Table 149. Organs were imaged at8 hours and the average total flux (photons/second) was measured for theliver, spleen, lung and kidney. A control for each organ was alsoanalyzed. The results are shown in Table 150. The peak signal for alldose levels was at 8 hours after administration. Also, distribution tothe various organs (liver, spleen, lung, and kidney) may be able to becontrolled by increasing or decreasing the LNP dose.

TABLE 149 Flux 0.5 mg/kg 0.05 mg/kg 0.005 mg/kg 0.0005 mg/kg Time PointFlux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) 2 hrs 1.23E+08 7.76E+067.66E+05 4.88E+05 8 hrs 1.05E+09 6.79E+07 2.75E+06 5.61E+05 24 hrs4.44E+07 1.00E+07 1.06E+06 5.71E+05 48 hrs 2.12E+07 4.27E+06 7.42E+054.84E+05 72 hrs 1.34E+07 5.84E+06 6.90E+05 4.38E+05 144 hrs 4.26E+062.25E+06 4.58E+05 3.99E+05

TABLE 150 Organ Flux Liver Spleen Lung Kidney Flux (p/s) Flux (p/s) Flux(p/s) Flux (p/s) 0.5 mg/kg 1.19E+08 9.66E+07 1.19E+06 1.85E+05 0.05mg/kg 1.10E+07 1.79E+06 7.23E+04 5.82E+04 0.005 mg/kg 3.58E+05 6.04E+041.33E+04 1.33E+04 0.0005 mg/kg 2.25E+04 1.88E+04 2.05E+04 1.65E+04Untreated 1.91E+04 1.66E+04 2.63E+04 2.14E+04

Example 98 Lipid Nanoparticle Subcutaneous Study

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) was formulated in alipid nanoparticle containing 50% DLin-KC2-DMA as described in Table151, 385% cholesterol, 10% DSPC and 1.5% PEG. The formulation wasadministered subcutaneously (S.C.) to Balb-C mice at a dose of 0.5mg/kg, 0.05 mg/kg or 0.005 mg/kg.

TABLE 151 DLin-KC2-DMA Formulation Formulation NPA-098-1 LipidDLin-KC2-DMA Lipid/mRNA ratio (wt/wt) 20:1 Mean Size 135 nm PDI: 0.08Zeta at pH 7.4 −0.6 mV Encaps. (RiboGr) 91%

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours, 24 hours, 48hours, 72 hours and 144 hours after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The lower limit of detection was about 3E+05p/s. The results of the imaging are shown in Table 152. Organs wereimaged at 8 hours and the average total flux (photons/second) wasmeasured for the liver, spleen, lung and kidney. A control for eachorgan was also analyzed. The results are shown in Table 153. The peaksignal for all dose levels was at 8 hours after administration. Also,distribution to the various organs (liver, spleen, lung, and kidney) maybe able to be controlled by increasing or decreasing the LNP dose. Athigh doses, the LNP formulations migrates outside of the subcutaneousinjection site, as high levels of luciferase expression are detected inthe liver, spleen, lung, and kidney.

TABLE 152 Flux 0.5 mg/kg 0.05 mg/kg 0.005 mg/kg Time Point Flux (p/s)Flux (p/s) Flux (p/s) 2 hrs 3.18E+07 7.46E+06 8.94E+05 8 hrs 5.15E+082.18E+08 1.34E+07 24 hrs 1.56E+08 5.30E+07 7.16E+06 48 hrs 5.22E+078.75E+06 9.06E+05 72 hrs 8.87E+06 1.50E+06 2.98E+05 144 hrs 4.55E+053.51E+05 2.87E+05

TABLE 153 Organ Flux Liver Spleen Lung Kidney Flux (p/s) Flux (p/s) Flux(p/s) Flux (p/s) 0.5 mg/kg 1.01E+07 7.43E+05 9.75E+04 1.75E+05 0.05mg/kg 1.61E+05 3.94E+04 4.04E+04 3.29E+04 0.005 mg/kg 2.84E+04 2.94E+042.42E+04 9.79E+04 Untreated 1.88E+04 1.02E+04 1.41E+04 9.20E+03

Example 99 Cationic Lipid Nanoparticle Subcutaneous Study

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine) is formulated in alipid nanoparticle containing 50% DLin-MC3-DMA, 38.5% cholesterol, 10%DSPC and 1.5% PEG. The formulation is administered subcutaneously (S.C.)to Balb-C mice at a dose of 0.5 mg/kg, 0.05 mg/kg or 0.005 mg/kg.

The mice are imaged at 2 hours, 8 hours, 24 hours, 48 hours, 72 hoursand 144 hours after dosing and the average total flux (photons/second)was measured for each route of administration and cationic lipidformulation. Organs are imaged at 8 hours and the average total flux(photons/second) is measured for the liver, spleen, lung and kidney. Acontrol for each organ is also analyzed.

Example 100 Luciferase Lipoplex Study

Lipoplexed luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1) fully modified with 5-methylcytosine and pseudouridine(5mC/pU), fully modified with 5-methylcytosine andN1-methyl-pseudouridine (5mC/N1mpU) or modified where 25% of thecytosines replaced with 5-methylcytosine and 25% of the uridinesreplaced with 2-thiouridine (5mC/s2U). The formulation was administeredintravenously (I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) toBalb-C mice at a dose of 0.10 mg/kg.

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 8 hours, 24 hours and 48 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and chemical modification. Thebackground signal was about 3.91E+05 p/s. The results of the imaging areshown in Table 154. Organs were imaged at 6 hours and the average totalflux (photons/second) was measured for the liver, spleen, lung andkidney. A control for each organ was also analyzed. The results areshown in Table 155.

TABLE 154 Flux 5mC/pU 5mC/N1mpU 5mC/s2U Route Time Point Flux (p/s) Flux(p/s) Flux (p/s) I.V. 8 hrs 5.76E+06 1.78E+06 1.88E+06 I.V. 24 hrs1.02E+06 7.13E+05 5.28E+05 I.V. 48 hrs 4.53E+05 3.76E+05 4.14E+05 I.M. 8hrs 1.90E+06 2.53E+06 1.29E+06 I.M. 24 hrs 9.33E+05 7.84E+05 6.48E+05I.M. 48 hrs 8.51E+05 6.59E+05 5.49E+05 S.C. 8 hrs 2.85E+06 6.48E+061.14E+06 S.C. 24 hrs 6.66E+05 7.15E+06 3.93E+05 S.C. 48 hrs 3.24E+053.20E+06 5.45E+05

TABLE 155 Organ Flux Liver Spleen Lung Kidney Route Chemistry Flux (p/s)Flux (p/s) Flux (p/s) Flux (p/s) Inj. Site Flux (p/s) I.V. 5mC/pU5.26E+05 2.04E+07 4.28E+06 1.77E+04 n/a I.V. 5mC/N1mpU 1.48E+05 5.00E+061.93E+06 1.77E+04 n/a I.V. 5mC/s2U 2.14E+04 3.29E+06 5.48E+05 2.16E+04n/a I.M. 5mC/pU 2.46E+04 1.38E+04 1.50E+04 1.44E+04 1.15E+06 I.M.5mC/N1mpU 1.72E+04 1.76E+04 1.99E+04 1.56E+04 1.20E+06 I.M. 5mC/s2U1.28E+04 1.36E+04 1.33E+04 1.07E+04 7.60E+05 S.C. 5mC/pU 1.55E+041.67E+04 1.45E+04 1.69E+04 4.46E+04 S.C. 5mC/N1mpU 1.20E+04 1.46E+041.38E+04 1.14E+04 8.29E+04 S.C. 5mC/s2U 1.22E+04 1.31E+04 1.45E+041.08E+04 5.62E+04 Untreated 2.59E+04 1.34E+04 1.26E+04 1.22E+04 n/a

Example 101 Cationic Lipid Formulation of Modified mRNA

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1)modified where 25% of the cytosines replaced with 5-methylcytosine and25% of the uridines replaced with 2-thiouridine (5mC/s2U) was formulatedin the cationic lipids described in Table 156. The formulations wereadministered intravenously (I.V.), intramuscularly (I.M.) orsubcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.

TABLE 156 Cationic Lipid Formulations Formulation NPA-130-1 NPA-131-1NPA-132-1 NPA-133-1 111612-C Lipid DLin-MC3- DLin-KC2- C12-200 DLinDMADODMA DMA DMA Lipid/mRNA 20:1 20:1 20:1 20:1 20:1 ratio (wt/wt) MeanSize 120 nm 105 nm 122 nm 105 nm 221.3 nm PDI: 0.10 PDI: 0.11 PDI: 0.13PDI: 0.14 PDI: 0.063 Zeta at pH 7.4 0.2 mV −0.6 mV −0.5 mV −0.3 mV −3.10mV Encaps. 100% 100% 93% 93% 60% (RiboGr)

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 3.31E+05 p/s. The results of the imaging areshown in Table 157. In Table 157, “NT” means not tested. Untreated miceshowed an average flux of 3.14E+05 at 2 hours, 3.33E+05 at 8 hours and3.46E+05 at 24 hours. Peak expression was seen for all three routestested at 8 hours. DLin-KC2-DMA has better expression than DLin-MC3-DMAand DODMA showed expression for all routes evaluated.

TABLE 157 Flux DLin- DLin- MC3- KC2- Time DMA DMA C12-200 DLinDMA DODMARoute Point Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V. 2 hrs 9.88E+06 6.98E+07 9.18E+06 3.98E+06 5.79E+06 I.V.  8 hrs 1.21E+071.23E+08 1.02E+07 5.98E+06 6.14E+06 I.V. 24 hrs 2.02E+06 1.05E+071.25E+06 1.35E+06 5.72E+05 I.M.  2 hrs 6.72E+05 3.66E+06 3.25E+067.34E+05 4.42E+05 I.M.  8 hrs 7.78E+06 2.85E+07 4.29E+06 2.22E+061.38E+05 I.M. 24 hrs 4.22E+05 8.79E+05 5.95E+05 8.48E+05 4.80E+05 S.C. 2 hrs 2.37E+06 4.77E+06 4.44E+06 1.07E+06 1.05E+06 S.C.  8 hrs 3.65E+071.17E+08 3.71E+06 9.33E+06 2.57E+06 S.C. 24 hrs 4.47E+06 1.28E+076.39E+05 8.89E+05 4.27E+05

Example 102 Formulation of 5-Methylcytosine and N1-Methyl-pseudouridineModified mRNA

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and N1-methyl-pseudouridine wasformulated in PBS (pH of 7.4). The formulations were administeredintramuscularly (I.M.) or subcutaneously (S.C.) to Balb-C mice at a doseof 2.5 mg/kg.

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 5 minutes, 30 minutes, 60minutes and 120 minutes after dosing and the average total flux(photons/second) was measured for each route of administration andcationic lipid formulation. The background flux was about 3.78E+05 p/s.The results of the imaging are shown in Table 158. Expression ofluciferase was already seen at 30 minutes with both routes of delivery.Peak expression from subcutaneous administration appears between 30 to60 minutes. Intramuscular expression was still increasing at 120minutes.

TABLE 158 Flux PBS (pH 7.4) Route Time Point Flux (p/s) I.M. 5 min4.38E+05 I.M. 30 min 1.09E+06 I.M. 60 min 1.18E+06 I.M. 120 min 2.86E+06S.C. 5 min 4.19E+05 S.C. 30 min 6.38E+06 S.C. 60 min 5.61E+06 S.C. 120min 2.66E+06

Example 103 Intramuscular and Subcutaneous Administration of ChemicallyModified mRNA

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) fully modified with N4-acetylcytidine, fully modified with5-methoxyuridine, fully modified with N4-acetylcytidine andN1-methyl-pseudouridine or fully modified 5-methylcytosine and5-methoxyuridine formulated in PBS (pH 7.4) was administered to Balb-Cmice intramuscularly or subcutaneously at a dose of 2.5 mg/kg. Twentyminutes prior to imaging, mice were injected intraperitoneally with aD-luciferin solution at 150 mg/kg. Animals were then anesthetized andimages were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hours.The average total flux (photons/second) for intramuscular administrationis shown in Table 159 and the average total flux (photons/second) forsubcutaneous administration is shown in Table 160. The background signalwas 3.84E+05 (p/s). The peak expression for intramuscular administrationwas seen between 24 and 48 hours for all chemistry and expression wasstill detected at 120 hours. For subcutaneous delivery the peakexpression was seen at 2-8 hours and expression was detected at 72hours.

TABLE 159 Intramuscular Administration 2 hours 8 hours 24 hours Flux(p/s) Flux (p/s) Flux (p/s) N4-acetylcytidine 1.32E+07 2.15E+07 4.01E+075-methoxyuridine 4.93E+06 1.80E+07 4.53E+07 N4-acetylcytidine/ 2.02E+071.93E+07 1.63E+08 N1-methyl-pseudouridine 5-methylcytosine/5- 6.79E+064.55E+07 3.44E+07 methoxyuridine

TABLE 160 Subcutaneous Administration 2 hours 8 hours 24 hours Flux(p/s) Flux (p/s) Flux (p/s) N4-acetylcytidine 3.07E+07 1.23E+07 1.28E+075-methoxyuridine 7.10E+06 9.38E+06 1.32E+07 N4-acetylcytidine/ 7.12E+063.07E+06 1.03E+07 N1-methyl-pseudouridine 5-methylcytosine/5- 7.15E+061.25E+07 1.11E+07 methoxyuridine

Example 104 In Vivo Study

Luciferase modified mRNA containing at least one chemical modificationis formulated as a lipid nanoparticle (LNP) using the syringe pumpmethod and characterized by particle size, zeta potential, andencapsulation.

As outlined in Table 161, the luciferase LNP formulation is administeredto Balb-C mice intramuscularly (I.M.), intravenously (I.V.) andsubcutaneously (S.C.). As a control luciferase modified RNA formulatedin PBS is administered intravenously to mice.

TABLE 161 Luciferase Formulations Concen- Injection Amount of trationVolume modified Dose Formulation Vehicle Route (mg/ml) (ul) RNA (ug)(mg/kg) Luc-LNP PBS S.C. 0.2000 50 10 0.5000 Luc-LNP PBS S.C. 0.0200 501 0.0500 Luc-LNP PBS S.C. 0.0020 50 0.1 0.0050 Luc-LNP PBS S.C. 0.000250 0.01 0.0005 Luc-LNP PBS I.V. 0.2000 50 10 0.5000 Luc-LNP PBS I.V.0.0200 50 1 0.0500 Luc-LNP PBS I.V. 0.0020 50 0.1 0.0050 Luc-LNP PBSI.V. 0.0002 50 0.01 0.0005 Luc-LNP PBS I.M. 0.2000 50 10 0.5000 Luc-LNPPBS I.M. 0.0200 50 1 0.0500 Luc-LNP PBS I.M. 0.0020 50 0.1 0.0050Luc-LNP PBS I.M. 0.0002 50 0.01 0.0005 Luc-PBS PBS I.V. 0.20  50 100.50 

The mice are imaged at 2, 8, 24, 48, 120 and 192 hours to determine thebioluminescence (measured as total flux (photons/second) of the entiremouse). At 8 hours or 192 hours the liver, spleen, kidney and injectionsite for subcutaneous and intramuscular administration are imaged todetermine the bioluminescence.

Example 105 Cationic Lipid Formulation Studies of Chemically ModifiedmRNA

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (5mC/pU), pseudouridine(pU) or N1-methyl-pseudouridine (N1mpU) was formulated in the cationiclipids described in Table 162. The formulations were administeredintravenously (I.V.), intramuscularly (I.M.) or subcutaneously (S.C.) toBalb-C mice at a dose of 0.05 mg/kg.

TABLE 162 Cationic Lipid Formulations Formulation NPA-137-1 NPA-134-1NPA-135-1 NPA-136-1 111612-A Lipid DLin-MC3- DLin-MC3- DLin-KC2- C12-200DODMA DMA DMA DMA Lipid/mRNA 20:1 20:1 20:1 20:1 20:1 ratio (wt/wt) MeanSize 111 nm 104 nm 95 nm 143 nm 223.2 nm PDI: 0.15 PDI: 0.13 PDI: 0.11PDI: 0.12 PDI: 0.142 Zeta at pH 7.4 −4.1 mV −1.9 mV −1.0 mV 0.2 mV −3.09mV Encaps. 97% 100% 100% 78% 64% (RiboGr) Chemistry pU N1mpU N1mpU N1mpU5mC/pU

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 4.11E+05 p/s. The results of the imaging areshown in Table 163. Peak expression was seen for all three routes testedat 8 hours.

TABLE 163 Flux DLin- DLin- DLin- MC3- MC3- KC2- DMA DMA DMA C12-200DODMA Time (pU) (N1mpU) (N1mpU) (N1mpU) (5mC/pU) Route Point Flux (p/s)Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V.  2 hrs 3.21E+081.24E+09 1.01E+09 9.00E+08 3.90E+07 I.V.  8 hrs 1.60E+09 3.22E+092.38E+09 1.11E+09 1.17E+07 I.V. 24 hrs 1.41E+08 3.68E+08 3.93E+088.06E+07 1.11E+07 I.M.  2 hrs 2.09E+07 3.29E+07 8.32E+07 9.43E+074.66E+06 I.M.  8 hrs 2.16E+08 6.14E+08 1.00E+09 8.77E+07 7.05E+06 I.M.24 hrs 1.23E+07 1.40E+08 5.09E+08 1.36E+07 1.14E+06 S.C.  2 hrs 2.32E+073.60E+07 2.14E+08 1.01E+08 3.11E+07 S.C.  8 hrs 5.55E+08 9.80E+084.93E+09 1.01E+09 8.04E+07 S.C. 24 hrs 1.81E+08 2.74E+08 2.12E+094.74E+07 1.34E+07

Example 106 Studies of Chemical Modified mRNA

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with N4-acetylcytidine (N4-acetyl), fully modified with5-methoxyuridine (5-meth), fully modified with N4-acetylcytidine andN1-methyl-pseudouridine (N4-acetyl/N1mpU) or fully modified with5-methylcytosine and 5-methoxyuridine (5mC/5-meth) was formulated inDLin-MC3-DMA as described in Table 164.

The formulations were administered intravenously (I.V.), intramuscularly(I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.

TABLE 164 Cationic Lipid Formulations Formulation NPA-141-1 NPA-142-1NPA-143-1 NPA-144-1 Lipid DLin-MC3- DLin-MC3- DLin-MC3- DLin-MC3- DMADMA DMA DMA Lipid/mRNA 20:1 20:1 20:1 20:1 ratio (wt/wt) Mean Size 138nm 116 nm 144 nm 131 nm PDI: 0.16 PDI: 0.15 PDI: 0.15 PDI: 0.15 Zeta atpH 7.4 −2.8 mV −2.8 mV −4.3 mV −5.0 mV Encaps. 97% 100% 75% 72% (RiboGr)Chemistry N4-acetyl 5meth N4-acetyl/ 5mC/5-meth N1mpU

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 6 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredfor each route of administration and cationic lipid formulation. Thebackground flux was about 2.70E+05 p/s. The results of the imaging areshown in Table 165.

TABLE 165 Flux N4-acetyl/ 5mC/5- N4-acetyl 5meth N1mpU meth Route TimePoint Flux (p/s) Flux (p/s) Flux (p/s) Flux (p/s) I.V. 2 hrs 9.17E+073.19E+06 4.21E+07 1.88E+06 I.V. 6 hrs 7.70E+08 9.28E+06 2.34E+087.75E+06 I.V. 24 hrs 6.84E+07 1.04E+06 3.55E+07 3.21E+06 I.M. 2 hrs8.59E+06 7.86E+05 5.30E+06 5.11E+05 I.M. 6 hrs 1.27E+08 8.88E+063.82E+07 3.17E+06 I.M. 24 hrs 4.46E+07 1.38E+06 2.00E+07 1.39E+06 S.C. 2hrs 1.83E+07 9.67E+05 4.45E+06 1.01E+06 S.C. 6 hrs 2.89E+08 1.78E+078.91E+07 1.29E+07 S.C. 24 hrs 6.09E+07 6.40E+06 2.08E+08 6.63E+06

Example 107 Lipid Nanoparticle Containing A Plurality of Modified mRNAs

EPO mRNA (mRNA sequence shown in SEQ ID NO: 1638; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and N1-methyl-pseudouridine), G-CSF mRNA(mRNA sequence shown in SEQ ID NO: 21438; polyA tail of approximately140 nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and N1-methyl-pseudouridine) and Factor IX mRNA (mRNAsequence shown in SEQ ID NO: 1622; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and N1-methyl-pseudouridine), is formulated inDLin-MC3-DMA as described in Table 166. The formulations areadministered intravenously (I.V.), intramuscularly (I.M.) orsubcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg. ControlLNP formulations containing only one mRNA are also admininistered at anequivalent dose.

TABLE 166 DLin-MC3-DMA Formulation Formulation NPA-157-1 LipidDLin-MC3-DMA Lipid/mRNA 20:1 ratio (wt/wt) Mean Size 89 nm PDI: 0.08Zeta at pH 7.4 1.1 mV Encaps. 97% (RiboGr)

Serum is collected from the mice at 8 hours, 24 hours, 72 hours and/or 7days after administration of the formulation. The serum is analyzed byELISA to determine the protein expression of EPO, G-CSF, and Factor IX.

Example 108 Cationic Lipid Formulation Studies of 5-Methylcytosine andN1-Methyl-pseudouridine Modified mRNA

EPO mRNA (mRNA sequence shown in SEQ ID NO: 1638; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and N1-methyl-pseudouridine) or G-CSFmRNA (mRNA sequence shown in SEQ ID NO: 21438; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and N1-methyl-pseudouridine) isformulated in DLin-MC3-DMA and DLin-KC2-DMA as described in Table 167.The formulations are administered intravenously (I.V), intramuscularly(I.M.) or subcutaneously (S.C.) to Balb-C mice at a dose of 0.05 mg/kg.

TABLE 167 DLin-MC3-DMA and DLin-KC2-DMA Formulations FormulationNPA-147-1 NPA-148-1 NPA-150-1 NPA-151-1 mRNA EPO EPO G-CSF G-CSF LipidDLin-MC3- DLin-KC2- DLin-MC3- DLin-KC2- DMA DMA DMA DMA Lipid/mRNA 20:120:1 20:1 20:1 ratio (wt/wt) Mean Size 117 nm 82 nm 119 nm 88 nm PDI:0.14 PDI: 0.08 PDI: 0.13 PDI: 0.08 Zeta at pH 7.4 −1.7 mV 0.6 mV 3.6 mV2.2 mV Encaps. 100% 96% 100% 100% (RiboGr)

Serum is collected from the mice at 8 hours, 24 hours, 72 hours and/or 7days after administration of the formulation. The serum is analyzed byELISA to determine the protein expression of EPO and G-CSF.

Example 109 In Vitro VEGF PBMC Study

500 ng of VEGF mRNA (mRNA sequence shown in SEQ ID NO: 1672 polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1)fully modified with 5-methylcytosine and pseudouridine (VEGF 5mC/pU),fully modified with 5-methylcytosine and N1-methyl-pseudouridine (VEGF5mC/N1mpU) or unmodified (VEGF unmod) was transfected with 0.4 uL ofLipofectamine 2000 into peripheral blood mononuclear cells (PBMC) fromthree normal blood donors (D1, D2, and D3). Cells were also untreatedfor each donor as a control. The supernatant was harvested and run byELISA 22 hours after transfection to determine the protein expressionand cytokine induction. The expression of VEGF and IFN-alpha inductionis shown in Table 168 and FIGS. 8A and 8B.

TABLE 168 Protein and Cytokine levels VEGF Expression IFN-alphaInduction (pg/ml) (pg/ml) D1 D2 D3 D1 D2 D3 VEGF unmod 2 0 0 5400 35374946 VEGF 5mC/pU 424 871 429 145 294 106 VEGF 5088 10331 6183 205 165 65mC/N1mpU

Example 110 In vitro expression of modified mRNA

HEK293 cells were transfected EPO modified mRNA (mRNA sequence shown inSEQ ID NO:1638; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine), HeLa cells were forward transfected with Transforminggrowth factor beta (TGF-beta) modified mRNA (mRNA sequence shown in SEQID NO: 1668; poly A tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) and HepG2 cells were transfected withbactericidal/permeability-increasing protein (rBPI-21) modified mRNA(SEQ ID NO: 21449; poly A tail of approximately 160 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytosineand pseudouridine) which had been complexed with Lipofectamine-2000 fromInvitrogen (Carlsbad, Calif.) at the concentrations shown in Table 169,170 and 171 using the procedures described herein. The proteinexpression was detected by ELISA and the protein (pg/ml) is also shownin Table 169, 170 and 171. In Table 169, “>” means greater than. ForTGF-beta a control of untreated cells and a mock transfection ofLipofectamine-2000 was also tested.

TABLE 169 EPO Protein Expression Amount 5 ng 1 ng 200 pg 40 pg 8 pg 1.6320 fg 64 fg Transfected pg Protein >2000 609.486 114.676 0 0 0 0 0(pg/ml)

TABLE 170 TGF-beta Protein Expression Amount Transfected 750 ng 250 ng83 ng Mock Untreated Protein 5058 4325 3210 2 0 (pg/ml)

TABLE 171 rBPI-21 Protein Expression Amount 2 ug 400 ng 80 ng 16 ng 3.2640 pg 128 26 pg Transfected ng pg Protein 20.683 9.269 4.768 0 0 0 0 0(pg/ml)

Example 111 Bicistronic modified mRNA

Human embryonic kidney epithelial (HEK293) were seeded on 96-well plates(Greiner Bio-one GmbH, Frickenhausen, Germany) HEK293 were seeded at adensity of 30,000 in 100 μl cell culture medium (DMEM, 10% FCS, adding 2mM L-Glutamine, 1 mM Sodiumpyruvate and 1× non-essential amino acids(Biochrom AG, Berlin, Germany) and 1.2 mg/ml Sodiumbicarbonate(Sigma-Aldrich, Munich, Germany)) 75 ng of the bi-cistronic modifiedmRNA (mCherry-2A-GFP) (SEQ ID NO: 21450; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine), mCherry modified mRNA (mRNA SEQ IDNO: 21439; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) or green fluorescent protein (GFP) modified mRNA (mRNAsequence shown in SEQ ID NO: 21448; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and pseudouridine) were added after seeding the cellsand incubated. A control of untreated cells was also evaluated.mCherry-2A-GFP refers to a modified mRNA sequence comprising the codingregion of mCherry, the 2A peptide and the coding region of GFP.

Cells were harvested by transferring the culture media supernatants to a96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg,Germany). Cells were trypsinized with ½ volume Trypsin/EDTA (BiochromAG, Berlin, Germany), pooled with respective supernatants and fixed byadding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5%formaldehyde (Merck, Darmstadt, Germany). Samples then were submitted toa flow cytometer measurement with a 532 nm excitation laser and the610/20 filter for PE-Texas Red in a LSRII cytometer (Beckton DickinsonGmbH, Heidelberg, Germany). The mean fluorescence intensity (MFI) of allevents is shown in Table 172. Cells transfected with the bi-cistronicmodified mRNA were able to express both mCherry and GFP.

TABLE 172 MFI of Modified mRNA Modified mRNA mCherry MFI GFP MFI mCherry17746 427 GFP 427 20019 mCherry-2A-GFP 5742 6783 Untreated 427 219

Example 112 Cationic Lipid Formulation Studies of 5-Methylcytosine andN1-Methyl-pseudouridine Modified mRNA to Produce an Antibody

Herceptin heavy chain (HC) mRNA (mRNA sequence shown in SEQ ID NO:21451; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andN1-methyl-pseudouridine) and Herceptin light chain (LC) (mRNA sequenceshown in SEQ ID NO: 21452; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1; fully modified with5-methylcytosine and N1-methyl-pseudouridine) are formulated inDLin-MC3-DMA and DLin-KC2-DMA as described in Table 173. Theformulations are administered intravenously (I.V) to Balb-C mice at adose of 0.500, 0.050, and 0.005 mg/kg.

TABLE 173 DLin-MC3-DMA and DLin-KC2-DMA Formulations FormulationNPA-158-1 NPA-159-1 Herceptin HC:LC 2:1 2:1 Ratio (wt/wt) LipidDLin-MC3-DMA DLin-KC2-DMA Lipid/Total mRNA 20:1 20:1 ratio (wt/wt) MeanSize 129 nm 100 nm PDI: 0.14 PDI: 0.10 Zeta at pH 7.4 0.9 mV 1.9 mVEncaps. (RiboGr) 100% 100%

Serum was collected from the mice at 8 hours, 24 hours, 72 hours and/or7 days after administration of the formulation. The serum was analyzedby ELISA to determine the protein expression of Herceptin.

Example 113 Directed SAR of Pseudouridine and N1-methyl-Pseudouridine

With the recent focus on the pyrimidine nucleoside pseudouridine, aseries of structure-activity studies were designed to investigate mRNAcontaining modifications to pseudouridine or N1-methyl-pseudourdine.

The study was designed to explore the effect of chain length, increasedlipophilicity, presence of ring structures, and alteration ofhydrophobic or hydrophilic interactions when modifications were made atthe N1 position, C6 position, the 2-position, the 4-position and on thephosphate backbone. Stability is also investigated.

To this end, modifications involving alkylation, cycloalkylation,alkyl-cycloalkylation, arylation, alkyl-arylation, alkylation moietieswith amino groups, alkylation moieties with carboxylic acid groups, andalkylation moieties containing amino acid charged moieties areinvestigated. The degree of alkylation is generally C₁-C₆. Examples ofthe chemistry modifications include those listed in Table 174 and Table175.

TABLE 174 Pseudouridine and N1-methylPseudouridine SAR CompoundNaturally Chemistry Modification # occuring N1-ModificationsN1-Ethyl-pseudo-UTP 1 N N1-Propyl-pseudo-UTP 2 NN1-iso-propyl-pseudo-UTP 3 N N1-(2,2,2-Trifluoroethyl)-pseudo-UTP 4 NN1-Cyclopropyl-pseudo-UTP 5 N N1-Cyclopropylmethyl-pseudo-UTP 6 NN1-Phenyl-pseudo-UTP 7 N N1-Benzyl-pseudo-UTP 8 NN1-Aminomethyl-pseudo-UTP 9 N P seudo-UTP-N1-2-ethanoic acid 10 N N1-(3-Amino-3-carboxypropyl)pseudo-UTP 11 NN1-Methyl-3-(3-amino-3-carboxypro- 12 Y pyl)pseudo-UTP C-6 Modifications6-Methyl-pseudo-UTP 13 N 6-Trifluoromethyl-pseudo-UTP 14 N6-Methoxy-pseudo-UTP 15 N 6-Phenyl-pseudo-UTP 16 N 6-Iodo-pseudo-UTP 17N 6-Bromo-pseudo-UTP 18 N 6-Chloro-pseudo-UTP 19 N 6-Fluoro-pseudo-UTP20 N 2- or 4-position Modifications 4-Thio-pseudo-UTP 21 N2-Thio-pseudo-UTP 22 N Phosphate backbone ModificationsAlpha-thio-pseudo-UTP 23 N N1-Me-alpha-thio-pseudo-UTP 24 N

TABLE 175 Pseudouridine and N1-methyl-Pseudouridine SAR CompoundNaturally Chemistry Modification # occuring N1-Methyl-pseudo-UTP  1 YN1-Butyl-pseudo-UTP  2 N N1-tert-Butyl-pseudo-UTP  3 NN1-Pentyl-pseudo-UTP  4 N N1-Hexyl-pseudo-UTP  5 NN1-Trifluoromethyl-pseudo-UTP  6 Y N1-Cyclobutyl-pseudo-UTP  7 NN1-Cyclopentyl-pseudo-UTP  8 N N1-Cyclohexyl-pseudo-UTP  9 NN1-Cycloheptyl-pseudo-UTP 10 N N1-Cyclooctyl-pseudo-UTP 11 NN1-Cyclobutylmethyl-pseudo-UTP 12 N N1-Cyclopentylmethyl-pseudo-UTP 13 NN1-Cyclohexylmethyl-pseudo-UTP 14 N N1-Cycloheptylmethyl-pseudo-UTP 15 NN1-Cyclooctylmethyl-pseudo-UTP 16 N N1-p-tolyl-pseudo-UTP 17 NN1-(2,4,6-Trimethyl-phenyl)pseudo-UTP 18 NN1-(4-Methoxy-phenyl)pseudo-UTP 19 N N1-(4-Amino-phenyl)pseudo-UTP 20 NN1(4-Nitro-phenyl)pseudo-UTP 21 N Pseudo-UTP-N1-p-benzoic acid 22 NN1-(4-Methyl-benzyl)pseudo-UTP 24 NN1-(2,4,6-Trimethyl-benzyl)pseudo-UTP 23 NN1-(4-Methoxy-benzyl)pseudo-UTP 25 N N1-(4-Amino-benzyl)pseudo-UTP 26 NN1-(4-Nitro-benzyl)pseudo-UTP 27 N Pseudo-UTP-N1-methyl-p-benzoic acid28 N N1-(2-Amino-ethyl)pseudo-UTP 29 N N1-(3-Amino-propyl)pseudo-UTP 30N N1-(4-Amino-butyl)pseudo-UTP 31 N N1-(5-Amino-pentyl)pseudo-UTP 32 NN1-(6-Amino-hexyl)pseudo-UTP 33 N Pseudo-UTP-N1-3-propionic acid 34 NPseudo-UTP-N1-4-butanoic acid 35 N Pseudo-UTP-N1-5-pentanoic acid 36 NPseudo-UTP-N1-6-hexanoic acid 37 N Pseudo-UTP-N1-7-heptanoic acid 38 NN1-(2-Amino-2-carboxyethyl)pseudo-UTP 39 NN1-(4-Amino-4-carboxybutyl)pseudo-UTP 40 N N3-Alkyl-pseudo-UTP 41 N6-Ethyl-pseudo-UTP 42 N 6-Propyl-pseudo-UTP 43 N 6-iso-Propyl-pseudo-UTP44 N 6-Butyl-pseudo-UTP 45 N 6-tert-Butyl-pseudo-UTP 46 N6-(2,2,2-Trifluoroethyl)-pseudo-UTP 47 N 6-Ethoxy-pseudo-UTP 48 N6-Trifluoromethoxy-pseudo-UTP 49 N 6-Phenyl-pseudo-UTP 50 N6-(Substituted-Phenyl)-pseudo-UTP 51 N 6-Cyano-pseudo-UTP 52 N6-Azido-pseudo-UTP 53 N 6-Amino-pseudo-UTP 54 N6-Ethylcarboxylate-pseudo-UTP  54b N 6-Hydroxy-pseudo-UTP 55 N6-Methylamino-pseudo-UTP  55b N 6-Dimethylamino-pseudo-UTP 57 N6-Hydroxyamino-pseudo-UTP 59 N 6-Formyl-pseudo-UTP 60 N6-(4-Morpholino)-pseudo-UTP 61 N 6-(4-Thiomorpholino)-pseudo-UTP 62 NN1-Me-4-thio-pseudo-UTP 63 N N1-Me-2-thio-pseudo-UTP 64 N1,6-Dimethyl-pseudo-UTP 65 N 1-Methyl-6-trifluoromethyl-pseudo-UTP 66 N1-Methyl-6-ethyl-pseudo-UTP 67 N 1-Methyl-6-propyl-pseudo-UTP 68 N1-Methyl-6-iso-propyl-pseudo-UTP 69 N 1-Methyl-6-butyl-pseudo-UTP 70 N1-Methyl-6-tert-butyl-pseudo-UTP 71 N1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP 72 N1-Methyl-6-iodo-pseudo-UTP 73 N 1-Methyl-6-bromo-pseudo-UTP 74 N1-Methyl-6-chloro-pseudo-UTP 75 N 1-Methyl-6-fluoro-pseudo-UTP 76 N1-Methyl-6-methoxy-pseudo-UTP 77 N 1-Methyl-6-ethoxy-pseudo-UTP 78 N1-Methyl-6-trifluoromethoxy-pseudo-UTP 79 N 1-Methyl-6-phenyl-pseudo-UTP80 N 1-Methyl-6-(substituted phenyl)pseudo-UTP 81 N1-Methyl-6-cyano-pseudo-UTP 82 N 1-Methyl-6-azido-pseudo-UTP 83 N1-Methyl-6-amino-pseudo-UTP 84 N 1-Methyl-6-ethylcarboxylate-pseudo-UTP85 N 1-Methyl-6-hydroxy-pseudo-UTP 86 N1-Methyl-6-methylamino-pseudo-UTP 87 N1-Methyl-6-dimethylamino-pseudo-UTP 88 N1-Methyl-6-hydroxyamino-pseudo-UTP 89 N 1-Methyl-6-formyl-pseudo-UTP 90N 1-Methyl-6-(4-morpholino)-pseudo-UTP 91 N1-Methyl-6-(4-thiomorpholino)-pseudo-UTP 92 N 1-Alkyl-6-vinyl-pseudo-UTP93 N 1-Alkyl-6-allyl-pseudo-UTP 94 N 1-Alkyl-6-homoallyl-pseudo-UTP 95 N1-Alkyl-6-ethynyl-pseudo-UTP 96 N 1-Alkyl-6-(2-propynyl)-pseudo-UTP 97 N1-Alkyl-6-(1-propynyl)-pseudo-UTP 98 N

Example 114 Incorporation of Naturally and Non-Naturally OccuringNucleosides

Naturally and non-naturally occurring nucleosides are incorporated intomRNA encoding a polypeptide of interest. Examples of these are given inTables 176 and 177. Certain commercially available nucleosidetriphosphates (NTPs) are investigated in the polynucleotides of theinvention. A selection of these are given in Table 176. The resultantmRNA are then examined for their ability to produce protein, inducecytokines, and/or produce a therapeutic outcome.

TABLE 176 Naturally and non-naturally occurring nucleosides CompoundNaturally Chemistry Modification # occuring N4-Methyl-Cytosine 1 YN4,N4-Dimethyl-2′-OMe-Cytosine 2 Y 5-Oxyacetic acid-methyl ester-Uridine3 Y N3-Methyl-pseudo-Uridine 4 Y 5-Hydroxymethyl-Cytosine 5 Y5-Trifluoromethyl-Cytosine 6 N 5-Trifluoromethyl-Uridine 7 N5-Methyl-amino-methyl-Uridine 8 Y 5-Carboxy-methyl-amino-methyl-Uridine9 Y 5-Carboxymethylaminomethyl-2′-OMe-Uridine 10 Y5-Carboxymethylaminomethyl-2-thio-Uridine 11 Y5-Methylaminomethyl-2-thio-Uridine 12 Y5-Methoxy-carbonyl-methyl-Uridine 13 Y5-Methoxy-carbonyl-methyl-2′-OMe-Uridine 14 Y 5-Oxyacetic acid-Uridine15 Y 3-(3-Amino-3-carboxypropyl)-Uridine 16 Y5-(carboxyhydroxymethyl)uridine methyl ester 17 Y5-(carboxyhydroxymethyl)uridine 18 Y

TABLE 177 Non-naturally occurring nucleoside triphosphates CompoundNaturally Chemistry Modification # occuring N1-Me-GTP 1 N2′-OMe-2-Amino-ATP 2 N 2′-OMe-pseudo-UTP 3 Y 2′-OMe-6-Me-UTP 4 N2′-Azido-2′-deoxy-ATP 5 N 2′-Azido-2′-deoxy-GTP 6 N2′-Azido-2′-deoxy-UTP 7 N 2′-Azido-2′-deoxy-CTP 8 N2′-Amino-2′-deoxy-ATP 9 N 2′-Amino-2′-deoxy-GTP 10 N2′-Amino-2′-deoxy-UTP 11 N 2′-Amino-2′-deoxy-CTP 12 N 2-Amino-ATP 13 N8-Aza-ATP 14 N Xanthosine-5′-TP 15 N 5-Bromo-CTP 16 N2′-F-5-Methyl-2′-deoxy-UTP 17 N 5-Aminoallyl-CTP 18 N2-Amino-riboside-TP 19 N

Example 115 Incorporation of Modifications to the Nucleobase andCarbohydrate (Sugar)

Naturally and non-naturally occurring nucleosides are incorporated intomRNA encoding a polypeptide of interest. Commercially availablenucleosides and NTPs having modifications to both the nucleobase andcarbohydrate (sugar) are examined for their ability to be incorporatedinto mRNA and to produce protein, induce cytokines, and/or produce atherapeutic outcome. Examples of these nucleosides are given in Tables178 and 179.

TABLE 178 Combination modifications Compound Chemistry Modification #5-iodo-2′-fluoro-deoxyuridine 1 5-iodo-cytidine 6 2′-bromo-deoxyuridine7 8-bromo-adenosine 8 8-bromo-guanosine 9 2,2′-anhydro-cytidinehydrochloride 10 2,2′-anhydro-uridine 11 2′-Azido-deoxyuridine 122-amino-adenosine 13 N4-Benzoyl-cytidine 14 N4-Amino-cytidine 152′-O-Methyl-N4-Acetyl-cytidine 16 2′Fluoro-N4-Acetyl-cytidine 172′Fluor-N4-Bz-cytidine 18 2′O-methyl-N4-Bz-cytidine 192′O-methyl-N6-Bz-deoxyadenosine 20 2′Fluoro-N6-Bz-deoxyadenosine 21N2-isobutyl-guanosine 22 2′Fluro-N2-isobutyl-guanosine 232′O-methyl-N2-isobutyl-guanosine 24

TABLE 179 Naturally occuring combinations Compound Naturally Name #occurring 5-Methoxycarbonylmethyl-2-thiouridine TP 1 Y5-Methylaminomethyl-2-thiouridine TP 2 Y 5-Crbamoylmethyluridine TP 3 Y5-Carbamoylmethyl-2′-O-methyluridine TP 4 Y1-Methyl-3-(3-amino-3-carboxypropyl) 5 Y pseudouridine TP5-Methylaminomethyl-2-selenouridine TP 6 Y 5-Carboxymethyluridine TP 7 Y5-Methyldihydrouridine TP 8 Y lysidine TP 9 Y 5-Taurinomethyluridine TP10 Y 5-Taurinomethyl-2-thiouridine TP 11 Y5-(iso-Pentenylaminomethyl)uridine TP 12 Y5-(iso-Pentenylaminomethyl)-2-thiouridine TP 13 Y5-(iso-Pentenylaminomethyl)-2′-O-methyl- 14 Y uridine TPN4-Acetyl-2′-O-methylcytidine TP 15 Y N4,2′-O-Dimethylcytidine TP 16 Y5-Formyl-2′-O-methylcytidine TP 17 Y 2′-O-Methylpseudouridine TP 18 Y2-Thio-2′-O-methyluridine TP 19 Y 3,2′-O-Dimethyluridine TP 20 Y

In the tables “UTP” stands for uridine triphosphate, “GTP” stands forguanosine triphosphate, “ATP” stands for adenosine triphosphate, “CTP”stands for cytosine triphosphate, “TP” stands for triphosphate and “Bz”stands for benzyl.

Example 116 Protein Production of Vascular Endothelial Growth Factor inHeLa Cells

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 250 ng ofvascular endothelial growth factor (VEGF) modified RNA (mRNA sequenceshown in SEQ ID NO: 1672; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1) with the chemical modificationdescribed in Table 180, was diluted in 10 ul final volume of OPTI-MEM(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul of lipofectamine 2000 was diluted to 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature. A second set of HeLa cells was also evaluated with the VEGFmRNA according to the procedure set out above. A control of untreatedcells and cells treated with just lipofecatamine 2000 was also analyzedfor the first set of HeLa cells.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing VEGF were collected and centrifuged at 10.000 rcf for 2minutes. The cleared supernatants were then analyzed with aVEGF-specific ELISA kit (R & D Systems, Minneapolis, Minn.) according tothe manufacturer instructions. All samples were diluted until thedetermined values were within the linear range of the ELISA standardcurve. The amount of protein produced is shown in Table 180.

These results demonstrate that VEGF fully modified with1-methylpseudouridine produced more protein in HeLa cells compared tothe other chemistry.

TABLE 180 Protein Production VEGF set 1 VEGF set 2 Sample (pg/ml)(pg/ml) 5-methylcytosine and pseudouridine 25,455 50,341 (5mC and pU)5-methylcytosine and 1-methylpsudouridine 5,795 8,523 (5mC and 1mpU) 25%of uridine modified with 2-thiouridine 27,045 27,045 and 25% of cytosinemodified with 5- methylcytosine (s2U and 5mC) Pseudouridine (pU) 216,023345,114 1-methylpseudouridine (1mpU) 32,614 38,523 Unmodified 409,318447,273 Lipofectamine 2000 (L2000) 0 N/A Untreated 0 N/A

Example 117 Comparison of Protein Production of Chemically ModifiedVascular Endothelial Growth Factor in HeLa Cells

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 250 ng ofvascular endothelial growth factor (VEGF) modified RNA (mRNA sequenceshown in SEQ ID NO: 1672; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1) with the chemical modificationdescribed in Table 181, were diluted in 10 ul final volume of OPTI-MEM(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul of lipofectamine 2000 was diluted to 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature. A control of untreated cells was also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing VEGF were collected and centrifuged at 10.000rcf for 2minutes. The cleared supernatants were then analyzed with aVEGF-specific ELISA kit (R & D Systems, Minneapolis, Minn.) according tothe manufacturer instructions. All samples were diluted until thedetermined values were within the linear range of the ELISA standardcurve. The amount of protein produced is shown in Table 181 and FIG. 9.

These results demonstrate that VEGF fully modified with1-methylpseudouridine produced more protein in HeLa cells compared tothe other chemistry.

TABLE 181 Protein Production VEGF Sample (pg/ml) Unmodified 121,1765-methylcytosine and 1-methylpsudouridine 760,000 (5mC and 1mpU)1-methylpseudouridine (1mpU) 1,393,824 Untreated 0

Example 118 Production of Vascular Endothelial Growth Factor Protein inMammals

Lipoplexed vascular endothelial growth factor (VEGF) mRNA (mRNA sequenceshown in SEQ ID NO: 1672; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1) unmodified, fully modified with5-methylcytosine and pseudouridine (5mC/pU), fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/1mpU), modified where25% of the cytosines replaced with 5-methylcytosine and 25% of theuridines replaced with 2-thiouridine (5mC/s2U), fully modified with1-methylpseudouridine (1 mpU) or fully modified with pseudouridine (pU).The formulation was administered intravenously to mice at a dose of 2 ugof mRNA per mouse. As a control, a group of mice were untreated. Serumfrom the mice is measured by specific VEGF ELISA 3 hours afteradministration for VEGF protein expression. The results are shown inTable 182 and FIG. 10.

TABLE 182 Protein Production VEGF Sample (pg/ml) Unmodified 345-methylcytosine and pseudouridine (5mC and pU) 40 5-methylcytosine and1-methylpsudouridine 11 (5mC and 1mpU) 25% of uridine modified with2-thiouridine and 53 25% of cytosine modified with 5-methylcytosine (s2Uand 5mC) Pseudouridine (pU) 86 1-methyl-pseudouridine (1mpU) 51Untreated 0

Example 119 Protein Production of Granulocyte Colony Stimulating Factorin HeLa Cells

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 250 ng ofgranulocyte colony stimulating factor (G-CSF) modified RNA (mRNAsequence shown in SEQ ID NO: 21438; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) with the chemicalmodification described in Table 183, was diluted in 10 ul final volumeof OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul of lipofectamine 2000 was diluted to 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature. A control of untreated cells and cells treated with justlipofecatamine 2000 was also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing G-CSF were collected and centrifuged at 10.000rcf for 2minutes. The cleared supernatants were then analyzed with aG-CSF-specific ELISA kit (R & D Systems, Minneapolis, Minn.) accordingto the manufacturer instructions. All samples were diluted until thedetermined values were within the linear range of the ELISA standardcurve. The amount of protein produced is shown in Table 183 and FIG. 11.

TABLE 183 Protein Production G-CSF Sample (pg/ml) 5-methylcytosine andpseudouridine (5mC and pU) 704,250 5-methylcytosine and1-methylpsudouridine 1,523,750 (5mC and 1mpU) 25% of uridine modifiedwith 2-thiouridine and 89,000 25% of cytosine modified with5-methylcytosine (s2U and 5mC) Pseudouridine (pU) 408,2501-methylpseudouridine (1mpU) 1,391,250 Lipofectamine 2000 (L2000) 0Untreated 0

Example 120 Production of Granulocyte Colony Stimulating Factor Proteinin Mammals

Granulocyte colony stimulating factor (G-CSF) mRNA (mRNA sequence shownin SEQ ID NO: 21438; polyA tail of approximately 160 nucleotides notshown in sequence; 5′ cap, Cap1) fully modified with 5-methylcytosineand pseudouridine (5mC/pU), fully modified with 5-methylcytosine and1-methylpseudouridine (5mC/lmpU), modified where 25% of the cytosinesreplaced with 5-methylcytosine and 25% of the uridines replaced with2-thiouridine (5mC/s2U), fully modified with 1-methylpseudouridine (1mpU) or fully modified with pseudouridine (pU). The formulation wasadministered intravenously to mice (CD1) (n=3) at a dose of 2 ug of mRNAand 2 ul of lipofectamine 2000 (L2000) per mouse. As a control, a groupof mice were untreated. Serum from the mice is measured by specificG-CSF ELISA 6 hours after administration for G-CSF protein expression.The results are shown in Table 184 and FIG. 12.

TABLE 184 Protein Production G-CSF Sample (pg/ml) 5-methylcytosine andpseudouridine (5mC and pU) 3,800 5-methylcytosine and1-methylpsudouridine (5mC and 1mpU) 22,868 25% of uridine modified with2-thiouridine and 25% of cytosine 1,386 modified with 5-methylcytosine(s2U and 5mC) Pseudouridine (pU) 7,579 1-methylpseudouridine (1mpU)25,882 Untreated 0

Example 121 Protein Production of Chemically Modified Factor IX in HeLaCell Supernatant

The day before transfection, 15,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul Eagle's minimal essential medium (EMEM) (supplemented with 10%fetal calf serum (FCS) and 1× Glutamax) per well in a 24-well cellculture plate (Corning, Manassas, Va.). The cells were grown at 37° C.in 5% CO₂ atmosphere overnight. Next day, 250 ng of factor IX modifiedRNA (mRNA sequence shown in SEQ ID NO: 1622; polyA tail of approximately140 nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (5mC/pU), fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/lmpU), modified where25% of the cytosines replaced with 5-methylcytosine and 25% of theuridines replaced with 2-thiouridine (s2U/5mC), fully modified with1-methylpseudouridine (1 mpU) or fully modified with pseudouridine (pU)were diluted in 10 ul final volume of OPTI-MEM® (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM®. After 5 minutes ofincubation at room temperature, both solutions were combined andincubated an additional 15 minute at room temperature. Then the 20 ulcombined solution was added to the 100 ul cell culture medium containingthe HeLa cells and incubated at room temperature. A control of untreatedwas also analyzed.

After an 18 hour incubation cell culture supernatants of cellsexpressing factor IX were collected by lysing cells withimmuno-precipitation (IP) buffer from Pierce Biotechnology (ThermoScientific, Rockford, Ill.) and centrifuged at 10.000rcf for 2 minutes.The cleared supernatants were diluted 1:2 (1 ml lysate/2 wells/24 wellplate) or 1:5 (1 ml lysate/5 wells/24 well plate) then analyzed with afactor IX-specific ELISA kit according to the manufacturer instructions.All samples were diluted until the determined values were within thelinear range of the ELISA standard curve. The amount of protein producedin both studies is shown in Table 185 and FIG. 13.

TABLE 185 Protein Production Factor IX Sample (ng/ml) 5-methylcytosineand pseudouridine (5mC/pU) 45.8 5-methylcytosine and1-methylpseudouridine 147.1 (5mC/1mpU) 25% of the cytosines replacedwith 5-methylcytosine 7.4 and 25% of the uridines replaced with2-thiouridine (s2U/5mC) Pseudouridine (pU) 17.0 1-methylpseudouridine(1mpU) 32.0 Untreated 0

Example 122 Protein Production of Apolipoprotein A-I in HeLa Cells

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul Eagle's minimal essential medium (EMEM) (supplemented with 10%fetal calf serum (FCS) and 1× Glutamax) per well in a 24-well cellculture plate (Corning, Manassas, Va.). The cells were grown at 37° C.in 5% CO₂ atmosphere overnight. Next day, 250 ng of apolipoprotein A-Iwild-type(APOA1wt) modified RNA (mRNA sequence shown in SEQ ID NO:21453; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andpseudouridine, apoliproprotein A1 Paris (APOA1 Paris) modified RNA (mRNAsequence shown in SEQ ID NO: 21454; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine or apoliproprotein A1 Milano (APOA1Milano) modified RNA (mRNA sequence shown in SEQ ID NO: 21455; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) fully modified with 5-methylcytosine and pseudouridine werediluted in 10 ul final volume of OPTI-MEM® (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM®. After 5 minutes ofincubation at room temperature, both solutions were combined andincubated an additional 15 minute at room temperature. Then the 20 ulcombined solution was added to the 100 ul cell culture medium containingthe HeLa cells and incubated at room temperature. A control of cellstreated with just lipofecatamine 2000 was also analyzed.

After an 18 hour incubation cell culture supernatants of cellsexpressing APOA1wt, APOA1 Paris or APOA1 Milano were collected by lysingcells with immuno-precipitation (IP) buffer from Pierce Biotechnology(Thermo Scientific, Rockford, Ill.) and centrifuged at 10.000rcf for 2minutes. The cleared supernatants were diluted 1:2 (1 ml lysate/2wells/24 well plate) or 1:5 (1 ml lysate/5 wells/24 well plate) thenanalyzed with an APOA1-specific ELISA kit according to the manufacturerinstructions. All samples were diluted until the determined values werewithin the linear range of the ELISA standard curve. The study wasrepeated and the amount of protein produced in both studies is shown inTable 186 and FIG. 14.

TABLE 186 Protein Production Protein Sample (ng/ml) APOA1wt 1:2 Dilution8 APOA1wt 1:2 Dilution Repeat 6 APOA1 Milano 1:2 Dilution 7 APOA1 Milano1:2 Dilution Repeat 7 APOA1 Paris 1:2 Dilution 10 APOA1 Paris 1:2Dilution Repeat 10 APOA1wt 1:5 Dilution 9 APOA1wt 1:5 Dilution Repeat 9APOA1 Milano 1:5 Dilution 8 APOA1 Milano 1:5 Dilution Repeat 6 APOA1Paris 1:5 Dilution 12 APOA1 Paris 1:5 Dilution Repeat 11 Control 1:2Dilution 0 Control 1:2 Dilution Repeat 0 Control 1:5 Dilution 0 Control1:5 Dilution Repeat 0

Example 123 Detection of Apolipoprotein A-I Wild-Type Protein: WesternBlot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 1250 ng of Apoliproprotein A-I (APOA1)wild-type mRNA (mRNA sequence shown in SEQ ID NO: 21453; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (5mC/pU), fullymodified with 5-methylcytosine and 1-methylpseudouridine (5mC/lmpU),modified where 25% of the cytosines replaced with 5-methylcytosine and25% of the uridines replaced with 2-thiouridine (s2U/5mC), fullymodified with 1-methylpseudouridine (1 mpU) or fully modified withpseudouridine (pU) was diluted in 250 ul final volume of OPTI-MEM®(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 5.5 ul were diluted in 250 ul final volume of OPTI-MEM®. After 5minutes of incubation at room temperature, both solutions were combinedand incubated an additional 15 minutes at room temperature. Then 500 ulof the combined solution was added to the 3 ml cell culture mediumcontaining the HeLa cells. The plates were then incubated as describedbefore.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4× SDS loading buffer (both from Life Technologies,Grand Island, N.Y.). Samples were heated at 95° C. for 5 minutes andloaded on the gel. Standard settings were chosen by the manufacturer,200V, 120 mA and maximum 25W. Run time was 60 minutes, but no longerthan running dye reaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. ApoA1 rabbit polyclonalantibody (Abcam, Cambridge, Mass.) against APOA1 were applied in 3 ml of5% BSA in 1× TBS solution at a 1:500 to 1:2000 dilution for 3 hours atroom temperature and gentle agitation on an orbital shaker. Membranesare washed 3 times with 1× TBS/0.1% Tween, 5 minutes each time withgentle agitation. Goat anti-rabbit HRP conjugate (Abcam, Cambridge,Mass.) was conjugated to horse radish peroxidase and binds to the ApoA1rabbit polyclonal antibody. The conjugated antibody was diluted of1:1000 to 1:5000 in 5% BSA in 1× TBS and incubated for 3 hours at roomtemperature. The Western Blot detection of APOA1 protein from APOA1wild-type mRNA containing various chemical modifications in HeLa celllysates is shown in FIG. 15.

Example 124 Detection of Apolipoprotein A-I Paris Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 1250 ng of Apoliproprotein A-I (APOA1)Paris mRNA (mRNA sequence shown in SEQ ID NO: 21454; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (5mC/pU), fullymodified with 5-methylcytosine and 1-methylpseudouridine (5mC/1 mpU),modified where 25% of the cytosines replaced with 5-methylcytosine and25% of the uridines replaced with 2-thiouridine (s2U/5mC), fullymodified with 1-methylpseudouridine (1 mpU) or fully modified withpseudouridine (pU) was diluted in 250 ul final volume of OPTI-MEM®(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 5.5 ul were diluted in 250 ul final volume of OPTI-MEM®. After 5minutes of incubation at room temperature, both solutions were combinedand incubated an additional 15 minutes at room temperature. Then 500 ulof the combined solution was added to the 3 ml cell culture mediumcontaining the HeLa cells. The plates were then incubated as describedbefore.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4× SDS loading buffer (both from Life Technologies,Grand Island, N.Y.). Samples were heated at 95° C. for 5 minutes andloaded on the gel. Standard settings were chosen by the manufacturer,200V, 120 mA and maximum 25W. Run time was 60 minutes, but no longerthan running dye reaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. ApoA1 rabbit polyclonalantibody (Abcam, Cambridge, Mass.) against APOA1 were applied in 3 ml of5% BSA in 1× TBS solution at a 1:500 to 1:2000 dilution for 3 hours atroom temperature and gentle agitation on an orbital shaker. Membranesare washed 3 times with 1× TBS/0.1% Tween, 5 minutes each time withgentle agitation. Goat anti-rabbit HRP conjugate (Abcam, Cambridge,Mass.) was conjugated to horse radish peroxidase and binds to the ApoA1rabbit polyclonal antibody. The conjugated antibody was diluted of1:1000 to 1:5000 in 5% BSA in 1×TBS and incubated for 3 hours at roomtemperature. The Western Blot detection of APOA1 protein from APOA1Paris mRNA containing various chemical modifications in HeLa celllysates is shown in FIG. 16.

Example 125 Detection of Apolipoprotein A-I Milano Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 1250 ng of Apoliproprotein A-I (APOA1)Milano mRNA (mRNA sequence shown in SEQ ID NO: 21455; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (5mC/pU), fullymodified with 5-methylcytosine and 1-methylpseudouridine (5mC/lmpU),modified where 25% of the cytosines replaced with 5-methylcytosine and25% of the uridines replaced with 2-thiouridine (s2U/5mC), fullymodified with 1-methylpseudouridine (1 mpU) or fully modified withpseudouridine (pU) was diluted in 250 ul final volume of OPTI-MEM®(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 5.5 ul were diluted in 250 ul final volume of OPTI-MEM®. After 5minutes of incubation at room temperature, both solutions were combinedand incubated an additional 15 minutes at room temperature. Then 500 ulof the combined solution was added to the 3 ml cell culture mediumcontaining the HeLa cells. The plates were then incubated as describedbefore.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4×SDS loading buffer (both from Life Technologies, GrandIsland, N.Y.). Samples were heated at 95° C. for 5 minutes and loaded onthe gel. Standard settings were chosen by the manufacturer, 200V, 120 mAand maximum 25W. Run time was 60 minutes, but no longer than running dyereaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. ApoA1 rabbit polyclonalantibody (Abcam, Cambridge, Mass.) against APOA1 were applied in 3 ml of5% BSA in 1× TBS solution at a 1:500 to 1:2000 dilution for 3 hours atroom temperature and gentle agitation on an orbital shaker. Membranesare washed 3 times with 1× TBS/0.1% Tween, 5 minutes each time withgentle agitation. Goat anti-rabbit HRP conjugate (Abcam, Cambridge,Mass.) was conjugated to horse radish peroxidase and binds to the ApoA1rabbit polyclonal antibody. The conjugated antibody was diluted of1:1000 to 1:5000 in 5% BSA in 1×TBS and incubated for 3 hours at roomtemperature. The Western Blot detection of APOA1 protein from APOA1Milano mRNA containing various chemical modifications in HeLa celllysates is shown in FIG. 17.

Example 126 Detection of Fibrinogen A Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO2atmosphere overnight. Next day, 1250 ng of Fibrinogen A (FGA) mRNA (mRNAsequence shown in SEQ ID NO: 21456; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/lmpU) was diluted in 250ul final volume of OPTI-MEM® (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 5.5 ul were diluted in 250 ul final volume ofOPTI-MEM®. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minutes at roomtemperature. Then 500 ul of the combined solution was added to the 3 mlcell culture medium containing the HeLa cells. The plates were thenincubated as described before.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4× SDS loading buffer (both from Life Technologies,Grand Island, N.Y.). Samples were heated at 95° C. for 5 minutes andloaded on the gel. Standard settings were chosen by the manufacturer,200V, 120 mA and maximum 25W. Run time was 60 minutes, but no longerthan running dye reaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. Fibrinogen A goatpolyclonal antibody (Abcam, Cambridge, Mass.) against fibrinogen A wereapplied in 3 ml of 5% BSA in 1×TBS solution at a 1:500 to 1:2000dilution for 3 hours at room temperature and gentle agitation on anorbital shaker. Membranes are washed 3 times with 1× TBS/0.1% Tween, 5minutes each time with gentle agitation. Donkey anti-goat HRP conjugate(Abcam, Cambridge, Mass.) was conjugated to horse radish peroxidase andbinds to the fibrinogen A goat polyclonal antibody. The conjugatedantibody was diluted of 1:1000 to 1:5000 in 5% BSA in 1× TBS andincubated for 3 hours at room temperature. As shown with the box in FIG.18 the Western Blot detection of fibrinogen A was close to the expectedsize of 95 kd.

Example 127 Protein Production of Chemically Modified Plasminogen inHeLa Cell Supernatant

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 250 ng ofplasminogen modified RNA (mRNA sequence shown in SEQ ID NO: 21457; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) fully modified with 5-methylcytosine and 1-methylpseudouridine,was diluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM. After 5 minutes of incubationat room temperature, both solutions were combined and incubated anadditional 15 minute at room temperature. Then the 20 ul combinedsolution was added to the 100 ul cell culture medium containing the HeLacells and incubated at room temperature. A control of erythropoietin(EPO) modified mRNA (mRNA sequence shown in SEQ ID NO: 1638; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1,fully modified with 5-methylcytosine and pseudouridine) and untreatedcells were also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing plasminogen was collected and centrifuged at 10.000rcf for 2minutes. The cleared supernatants were then analyzed with aplasminogen-specific ELISA kit (R & D Systems, Minneapolis, Minn.)according to the manufacturer instructions. All samples were diluteduntil the determined values were within the linear range of the ELISAstandard curve. The amount of protein produced is shown in Table 187 andFIG. 19.

TABLE 187 Protein Production Plasminogen Sample (ng/ml) Plasminogen 10EPO 1.1 Untreated 0.8

Example 128 Detection of Plasminogen Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO2atmosphere overnight. Next day, 1250 ng of Plasminogen mRNA (mRNAsequence shown in SEQ ID NO: 21457; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/lmpU) was diluted in 250ul final volume of OPTI-MEM® (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 5.5 ul were diluted in 250 ul final volume ofOPTI-MEM®. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minutes at roomtemperature. Then 500 ul of the combined solution was added to the 3 mlcell culture medium containing the HeLa cells. The plates were thenincubated as described before.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4×SDS loading buffer (both from Life Technologies, GrandIsland, N.Y.). Samples were heated at 95° C. for 5 minutes and loaded onthe gel. Standard settings were chosen by the manufacturer, 200V, 120 mAand maximum 25W. Run time was 60 minutes, but no longer than running dyereaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. Plasminogen goatpolyclonal antibody (Abcam, Cambridge, Mass.) against plasminogen wereapplied in 3 ml of 5% BSA in 1×TBS solution at a 1:500 to 1:2000dilution for 3 hours at room temperature and gentle agitation on anorbital shaker. Membranes are washed 3 times with 1× TBS/0.1% Tween, 5minutes each time with gentle agitation. Donkey anti-goat HRP conjugate(Abcam, Cambridge, Mass.) was conjugated to horse radish peroxidase andbinds to the plasminogen goat polyclonal antibody. The conjugatedantibody was diluted of 1:1000 to 1:5000 in 5% BSA in 1× TBS andincubated for 3 hours at room temperature. FIG. 20 shows thatPlasminogen was detected close to the expected size of 95 kd.

Example 129 Detection of Galactose-1-phosphate UridylyltransferaseProtein Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 1250 ng of galactose-1-phosphateuridylyltransferase (GALT) mRNA (mRNA sequence shown in SEQ ID NO:21458; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andpseudouridine (5mC/pU) was diluted in 250 ul final volume of OPTI-MEM®(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 5.5 ul were diluted in 250 ul final volume of OPTI-MEM®. After 5minutes of incubation at room temperature, both solutions were combinedand incubated an additional 15 minutes at room temperature. Then 500 ulof the combined solution was added to the 3 ml cell culture mediumcontaining the HeLa cells. The plates were then incubated as describedbefore.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4× SDS loading buffer (both from Life Technologies,Grand Island, N.Y.). Samples were heated at 95° C. for 5 minutes andloaded on the gel. Standard settings were chosen by the manufacturer,200V, 120 mA and maximum 25W. Run time was 60 minutes, but no longerthan running dye reaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. GALT mouse monoclonalantibody (Novus biological, Littleton Colo.) against GALT were appliedin 3 ml of 5% BSA in 1× TBS solution at a 1:500 to 1:2000 dilution for 3hours at room temperature and gentle agitation on an orbital shaker.Membranes are washed 3 times with 1× TBS/0.1% Tween, 5 minutes each timewith gentle agitation. Donkey anti-mouse HRP conjugate (Abcam,Cambridge, Mass.) was conjugated to horse radish peroxidase and binds tothe GALT mouse monoclonal antibody. The conjugated antibody was dilutedof 1:1000 to 1:5000 in 5% BSA in 1× TBS and incubated for 3 hours atroom temperature. As shown with the box in FIG. 21, the Western Blotdetection of GALT was about 42 kd.

Example 130 Detection of Argininosuccinase Acid Lyase Protein: WesternBlot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 1250 ng of argininosuccinase acid lyase(ASL) mRNA (mRNA sequence shown in SEQ ID NO: 21459; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (5mC/pU) was diluted in250 ul final volume of OPTI-MEM® (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 5.5 ul were diluted in 250 ul final volume ofOPTI-MEM®. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minutes at roomtemperature. Then 500 ul of the combined solution was added to the 3 mlcell culture medium containing the HeLa cells. The plates were thenincubated as described before.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4×SDS loading buffer (both from Life Technologies, GrandIsland, N.Y.). Samples were heated at 95° C. for 5 minutes and loaded onthe gel. Standard settings were chosen by the manufacturer, 200V, 120 mAand maximum 25W. Run time was 60 minutes, but no longer than running dyereaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. ASL mouse monoclonalantibody (Novus biological, Littleton, Colo.) against ASL were appliedin 3 ml of 5% BSA in 1× TBS solution at a 1:500 to 1:2000 dilution for 3hours at room temperature and gentle agitation on an orbital shaker.Membranes are washed 3 times with 1× TBS/0.1% Tween, 5 minutes each timewith gentle agitation. Donkey anti-mouse HRP conjugate (Abcam,Cambridge, Mass.) was conjugated to horse radish peroxidase and binds tothe ASL mouse monoclonal antibody. The conjugated antibody was dilutedof 1:1000 to 1:5000 in 5% BSA in 1× TBS and incubated for 3 hours atroom temperature. As shown with the box in FIG. 22 the Western Blotdetection of ASL was about 50 kd.

Example 131 Detection of Tyrosine Aminotransferase Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 1250 ng of tyrosine aminotransferase(TAT) mRNA (mRNA sequence shown in SEQ ID NO: 21460; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and 1-methylpseudouridine (5mC/lmpU) wasdiluted in 250 ul final volume of OPTI-MEM® (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 5.5 ul were diluted in 250 ul finalvolume of OPTI-MEM®. After 5 minutes of incubation at room temperature,both solutions were combined and incubated an additional 15 minutes atroom temperature. Then 500 ul of the combined solution was added to the3 ml cell culture medium containing the HeLa cells. The plates were thenincubated as described before.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26ul, 4 ul of 10× reducingagent and 10 ul 4×SDS loading buffer (both from Life Technologies, GrandIsland, N.Y.). Samples were heated at 95° C. for 5 minutes and loaded onthe gel. Standard settings were chosen by the manufacturer, 200V, 120 mAand maximum 25W. Run time was 60 minutes, but no longer than running dyereaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. TAT rabbit polyclonalantibody (Novus biologicals) against TAT were applied in 3 ml of 5% BSAin 1× TBS solution at a 1:500 to 1:2000 dilution for 3 hours at roomtemperature and gentle agitation on an orbital shaker. Membranes arewashed 3 times with 1× TBS/0.1% Tween, 5 minutes each time with gentleagitation. Goat anti-rabbit HRP conjugate (Abcam, Cambridge, Mass.) wasconjugated to horse radish peroxidase and binds to the TAT rabbitpolyclonal antibody. The conjugated antibody was diluted of 1:1000 to1:5000 in 5% BSA in 1× TBS and incubated for 3 hours at roomtemperature. As shown with the box in FIG. 23 the Western Blot detectionof TAT was about 50 kd with the chemoluminescence band shown in white.

Example 132 Detection of 1,4-Alpha-Glucan-Branching Enzyme Protein:Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 1250 ng of 1,4-alpha-glucan-branchingenzyme (GBE1) mRNA (mRNA sequence shown in SEQ ID NO: 21461; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1)fully modified with 5-methylcytosine and 1-methylpseudouridine(5mC/lmpU) was diluted in 250 ul final volume of OPTI-MEM®(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 5.5 ul were diluted in 250 ul final volume of OPTI-MEM®. After 5minutes of incubation at room temperature, both solutions were combinedand incubated an additional 15 minutes at room temperature. Then 500 ulof the combined solution was added to the 3 ml cell culture mediumcontaining the HeLa cells. The plates were then incubated as describedbefore.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4×SDS loading buffer (both from Life Technologies, GrandIsland, N.Y.). Samples were heated at 95° C. for 5 minutes and loaded onthe gel. Standard settings were chosen by the manufacturer, 200V, 120 mAand maximum 25W. Run time was 60 minutes, but no longer than running dyereaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. GBE1 rabbit polyclonalantibody (Novus biological, Littleton, Colo.)) against GBE1 were appliedin 3 ml of 5% BSA in 1× TBS solution at a 1:500 to 1:2000 dilution for 3hours at room temperature and gentle agitation on an orbital shaker.Membranes are washed 3 times with 1× TBS/0.1% Tween, 5 minutes each timewith gentle agitation. Goat anti-rabbit HRP conjugate (Abcam, Cambridge,Mass.) was conjugated to horse radish peroxidase and binds to the GBE1rabbit polyclonal antibody. The conjugated antibody was diluted of1:1000 to 1:5000 in 5% BSA in 1× TBS and incubated for 3 hours at roomtemperature. As shown with the box in FIG. 24 the Western Blot detectionof GBE1 was about the expected size of 70 kd.

Example 133 Protein Production of Prothrombin in HeLa Cell Supernatant

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 250 ng ofprothrombin modified RNA (mRNA sequence shown in SEQ ID NO: 21462; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) fully modified with 5-methylcytosine and 1-methylpseudouridine,was diluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM. After 5 minutes of incubationat room temperature, both solutions were combined and incubated anadditional 15 minute at room temperature. Then the 20 ul combinedsolution was added to the 100 ul cell culture medium containing the HeLacells and incubated at room temperature. A control of erythropoietin(EPO) modified mRNA (mRNA sequence shown in SEQ ID NO: 1638; polyA tailof approximately 160 nucleotides not shown in sequence; 5′ cap, Cap1,fully modified with 5-methylcytosine and pseudouridine) and untreatedcells were also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing plasminogen was collected and centrifuged at 10.000rcf for 2minutes. The cleared supernatants were then analyzed with aprothrombin-specific ELISA kit (R & D Systems, Minneapolis, Minn.)according to the manufacturer instructions. All samples were diluteduntil the determined values were within the linear range of the ELISAstandard curve. The amount of protein produced is shown in Table 188 andFIG. 25.

TABLE 188 Protein Production Prothrombin Sample (ng/ml) Prothrombin 31EPO 2.5 Untreated 2

Example 134 Protein Production of Chemically Modified Prothrombin inHeLa Cell Supernatant

The day before transfection, 15,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100ul Eagle's minimal essential medium (EMEM) (supplemented with 10%fetal calf serum (FCS) and 1× Glutamax) per well in a 24-well cellculture plate (Corning, Manassas, Va.). The cells were grown at 37° C.in 5% CO₂ atmosphere overnight. Next day, 250 ng of prothrombin modifiedRNA (mRNA sequence shown in SEQ ID NO: 21462; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (5mC/pU), fullymodified with 5-methylcytosine and 1-methylpseudouridine (5mC/lmpU),modified where 25% of the cytosines replaced with 5-methylcytosine and25% of the uridines replaced with 2-thiouridine (s2U/5mC), fullymodified with 1-methylpseudouridine (1 mpU) or fully modified withpseudouridine (pU) were diluted in 10 ul final volume of OPTI-MEM®(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul of lipofectamine 2000 was diluted to 10 ul final volume ofOPTI-MEM®. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature. A control of untreated was also analyzed.

After an 18 hour incubation cell culture supernatants of cellsexpressing prothrombin were collected by lysing cells withimmuno-precipitation (IP) buffer from Pierce Biotechnology (ThermoScientific, Rockford, Ill.) and centrifuged at 10.000 rcf for 2 minutes.The cleared supernatants were diluted 1:2 (1 ml lysate/2 wells/24 wellplate) or 1:5 (1 ml lysate/5 wells/24 well plate) then analyzed with aprothrombin-specific ELISA kit according to the manufacturerinstructions. All samples were diluted until the determined values werewithin the linear range of the ELISA standard curve. The amount ofprotein produced in both studies is shown in Table 189 and FIG. 26.

TABLE 189 Protein Production Prothrombin Sample (ng/ml) 5-methylcytosineand pseudouridine (5mC/pU) 68 5-methylcytosine and 1-methylpseudouridine(5mC/1mpU) 210 25% of the cytosines replaced with 5-methylcytosine and25 25% of the uridines replaced with 2-thiouridine (s2U/5mC)1-methylpseudouridine (1mpU) 277 pseudouridine (pU) 30 Untreated 7.5

Example 135 Detection of Ceruloplasmin Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO2atmosphere overnight. Next day, 1250 ng of ceruloplasmin (CP or CLP)mRNA (mRNA sequence shown in SEQ ID NO: 1621; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and 1-methylpseudouridine (5mC/lmpU) wasdiluted in 250 ul final volume of OPTI-MEM® (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 5.5 ul were diluted in 250 ul finalvolume of OPTI-MEM®. After 5 minutes of incubation at room temperature,both solutions were combined and incubated an additional 15 minutes atroom temperature. Then 500 ul of the combined solution was added to the3 ml cell culture medium containing the HeLa cells. The plates were thenincubated as described before.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4×SDS loading buffer (both from Life Technologies, GrandIsland, N.Y.). Samples were heated at 95° C. for 5 minutes and loaded onthe gel. Standard settings were chosen by the manufacturer, 200V, 120 mAand maximum 25W. Run time was 60 minutes, but no longer than running dyereaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. Ceruloplasmin rabbitpolyclonal antibody (Novus biological, Littleton, Colo.) against CPproteins were applied in 3 ml of 5% BSA in lx TBS solution at a 1:500 to1:2000 dilution for 3 hours at room temperature and gentle agitation onan orbital shaker. Membranes are washed 3 times with 1× TBS/0.1% Tween,5 minutes each time with gentle agitation. Goat anti-rabbit HRPconjugate (Abcam, Cambridge, Mass.) was conjugated to horse radishperoxidase and binds to the Ceruloplasmin rabbit polyclonal antibody.The conjugated antibody was diluted of 1:1000 to 1:5000 in 5% BSA in 1×TBS and incubated for 3 hours at room temperature. As shown with the boxin FIG. 27 the Western Blot detection of ceruloplasmin (CLP) was about148 kd.

Example 136 Protein Production of Transforming Growth Factor Beta 1 inHeLa Cell Supernatant

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 83 ng, 250 ngor 750 ng of transforming growth factor beta 1 (TGF-beta1) modified RNA(mRNA sequence shown in SEQ ID NO: 1668; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine, was diluted in 1 0 ul final volumeof OPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul of lipofectamine 2000 was diluted to 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature. A control of lipofecatmine 2000 treated cells (L2000) anduntreated cells was also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing TGF-beta1 was collected and centrifuged at 10.000 rcf for 2minutes. The cleared supernatants were then analyzed with aTGF-beta1-specific ELISA kit (R & D Systems, Minneapolis, Minn.)according to the manufacturer instructions. All samples were diluteduntil the determined values were within the linear range of the ELISAstandard curve. The amount of protein produced is shown in Table 190 andFIG. 28.

TABLE 190 Protein Production TGF-beta1 Sample (pg/ml) TGF-beta1 (83 ng)3210 TGF-beta1 (250 ng) 4325 TGF-beta1 (750 ng) 5058 L2000 2 Untreated 0

Example 137 Detection of Ornithine Transcarbamoylase Protein: WesternBlot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 1250 ng of ornithine transcarbamoylase(OTC) mRNA (mRNA sequence shown in SEQ ID NO: 1659; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (5mC/pU) was diluted in250 ul final volume of OPTI-MEM® (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 5.5 ul were diluted in 250 ul final volume ofOPTI-MEM®. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minutes at roomtemperature. Then 500 ul of the combined solution was added to the 3 mlcell culture medium containing the HeLa cells. The plates were thenincubated as described before.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitationassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4× SDS loading buffer (both from Life Technologies,Grand Island, N.Y.). Samples were heated at 95° C. for 5 minutes andloaded on the gel. Standard settings were chosen by the manufacturer,200V, 120 mA and maximum 25W. Run time was 60 minutes, but no longerthan running dye reaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. OTC rabbit polyclonalantibody (Novus biological, Littleton, Colo.) against OTC proteins wereapplied in 3 ml of 5% BSA in lx TBS solution at a 1:500 to 1:2000dilution for 3 hours at room temperature and gentle agitation on anorbital shaker. Membranes are washed 3 times with 1× TBS/0.1% Tween, 5minutes each time with gentle agitation. Goat anti-rabbit HRP conjugate(Abcam, Cambridge, Mass.) was conjugated to horse radish peroxidase andbinds to the OTC rabbit polyclonal antibody. The conjugated antibody wasdiluted of 1:1000 to 1:5000 in 5% BSA in 1× TBS and incubated for 3hours at room temperature. As shown with the box in FIG. 29 the WesternBlot detection of OTC was about the expected size of 40 kd.

Example 138 LDLR In Vivo Study in Mammals

Low density lipoprotein (LDL) receptor (LDLR) mRNA (mRNA shown in SEQ IDNO: 21463; fully modified with 5-methylcytosine and pseudouridine; 5′cap, Cap1; polyA tail of 160 nucleotides not shown in the sequence) wascomplexed with Lipofectamine 2000 by mixing 8.0 μg mRNA with Dulbecco'smodified Eagle's medium (DMEM) to a final volume of 0.2 mL.

Lipofectamine 2000 was diluted 12.5-fold with DMEM and mixed with anequivalent volume of the diluted LDL receptor mRNA solution. The sampleswere incubated 5 minutes at room temperature and a 0.1 mL volume of thecomplexed mRNA mixture was injected into the tail vein of each of threeC57BL/6 mice. Each animal received a total dose of 2.0 μg of LDLreceptor mRNA. After 6 hours, the animals were sacrificed and thespleens were removed. Splenocytes were isolated according to standardprocedures (with no prior lysis of red blood cells) and stained withequivalent amounts of either IgG specific for the human LDL receptor ornon-immune IgG as a control.

The expression of the LDL receptors was assessed by flow cytometry withgating on the CD11b+ splenocyte population. As shown in FIG. 30, theexpression of LDL receptors in the CD11b+ splenocyte population in vivowas evident in each of three separate mice by the presence of rightwardshifted peaks that were stained with LDL receptor IgG (LDLR IgG) ascompared to cells stained with non-immune IgG (non-immune IgG).

For mice treated with Lipofectamine alone, no LDL receptor specific peakwas observed and staining was similar to that observed with non-immuneIgG.

Example 139 Modified UGT1A1 mRNA Study

HEK293 cells were seeded at density of 500,000 cells per 6-well plate inDMEM containing 10% fetal bovine serum and cells were grown overnightaccording to standard procedures. The next day 800 ng of UDPglucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) mRNA (mRNAshown in SEQ ID NO: 21464; fully modified with 5-methylcytosine andpseudouridine; 5′ cap, Cap1; polyA tail of 160 nucleotides not shown inthe sequence) was diluted into 0.3 mL of Optimum buffer, a 4.0 μL sampleof Lipofectamine 2000 was also diluted to 0.3 mL of Optimum buffer, andthe mRNA was complexed to Lipofectamine 2000 by mixing the twosolutions.

After incubation at room temperature for 15 minutes, cells weretransfected with the UGT1A1 mRNA/lipofectamine 2000 mixture. Afterincubation for 18 hours at 37° C., the media were aspirated and thecells were washed with PBS. Cells were harvested by scraping with a celllifter and the cells were centrifuged at 3000 rpm for 3 minutes. Thecell pellet was washed with PBS, centrifuged as described and the cellpellets were lysed by the addition of 0.25 mL of RIPA buffersupplemented with a protease inhibitor cocktail. Cell extracts werecentrifuged for 10 minutes at 12,210 rpm and the supernatant fractions(lysates) were frozen at −80° C.

Western blotting was conducted using the Simple Simon Western capillaryelectrophoresis apparatus using antibodies specific for human UGT1A1. A0.005 mg sample of lysate was run in triplicate. As shown in FIG. 31,UGT1A1 was detected as a single band of approximately 60 kDa. Incontrast, no bands were detected using lysates from control cellstransfected with an unrelated mRNA.

Example 140 In vivo expression of LDLR in mice

LDLR −/− mice are used to test the in vivo expression of LDLR mmRNA. LDLmmRNA is administered to LDLR −/− mice by injection. Tissues from themice are examined for LDLR expression. Western blot analysis of mousetissues is carried out to look for LDLR protein expression as a resultof LDLR mmRNA administration. Real time RT-PCR is carried out on mousetissues to look for LDLR gene expression.

Example 141 Production of LDLR in Mammals

Ornithine transcarbamoylase (OTC) is a mitochondrial protein encoded inthe nuclear genome. Defects in OTC are characterized by toxicaccumulation of ammonia and lead to urea cycle disorders.

Modified mRNA encoding OTC are administered to the mice described inExample 140. Serum, tissue and/or organs are collected at various timepoints in order to determine protein expression.

Further studies where LDLR modified mRNA are administered to the mice ofExample 140 are also conducted. Serum, tissue and/or organs arecollected at various time points in order to determine proteinexpression.

Example 142 Production of Chemically Modified Factor XI in HEK293 Cells

Human embryonic kidney epithelial (HEK293) (LGC standards GmbH, Wesel,Germany) are seeded on 24-well plates (Greiner Bio-one GmbH,Frickenhausen, Germany) precoated with collagen type1. HEK293 are seededat a density of about 100,000 cells per well in 100 μl cell culturemedium. Formulations containing 350 ng or 750 of Factor XI mRNA (modRNAFXI) (mRNA sequence shown in SEQ ID NO: 1625; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) areadded directly after seeding the cells and incubated. A control ofuntreated cells was also evaluated.

Cells are harvested by transferring the culture media supernatants to a96-well Pro-Bind U-bottom plate (Beckton Dickinson GmbH, Heidelberg,Germany). Cells are trypsinized with ½ volume Trypsin/EDTA (Biochrom AG,Berlin, Germany), pooled with respective supernatants and fixed byadding one volume PBS/2% FCS (both Biochrom AG, Berlin, Germany)/0.5%formaldehyde (Merck, Darmstadt, Germany). After a 12 hour incubation,cell culture supernatants of cells expressing Factor XI were collectedand centrifuged at 10.000 rcf for 2 minutes. The cleared supernatantswere then analyzed with a Factor XI-specific ELISA kit (InnovativeResearch, Novi, Mich.) according to the manufacturer instructions. Theamount of protein produced is shown in FIG. 32.

Example 143 Detection of Acmaporin-5 Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 1250 ng of aquaporin-5 mRNA (mRNAsequence shown in SEQ ID NO: 1617; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (5mC/pU), fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/lmpU), 25% of uridinemodified with 2-thiouridine and 25% of cytosine modified with5-methylcytosine (s2U and 5mC), fully modified with pseudouridine (pU)or fully modified with 1-methylpseudouridine (1 mpU) was diluted in 250ul final volume of OPTI-MEM® (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 5.5 ul were diluted in 250 ul final volume ofOPTI-MEM®. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minutes at roomtemperature. Then 500 ul of the combined solution was added to the 3 mlcell culture medium containing the HeLa cells. The plates were thenincubated as described before. A control of untreated cells was alsoevaluated.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitationassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4× SDS loading buffer (both from Life Technologies,Grand Island, N.Y.). Samples were heated at 95° C. for 5 minutes andloaded on the gel. Standard settings were chosen by the manufacturer,200V, 120 mA and maximum 25W. Run time was 60 minutes, but no longerthan running dye reaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1x tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. Aquaporin-5 rabbitpolyclonal antibody (Abcam, Cambridge, Mass.) against aquaporin-5proteins were applied in 3 ml of 5% BSA in 1× TBS solution at a 1:500 to1:2000 dilution for 3 hours at room temperature and gentle agitation onan orbital shaker. Membranes are washed 3 times with 1× TBS/0.1% Tween,5 minutes each time with gentle agitation. Goat anti-rabbit conjugate(Abcam, Cambridge, Mass.) was conjugated to horse radish peroxidase andbinds to the Aquaporin-5 rabbit polyclonal antibody. The conjugatedantibody was diluted of 1:1000 to 1:5000 in 5% BSA in 1× TBS andincubated for 3 hours at room temperature. As shown with the box in FIG.33 the Western Blot detected protein in each of the chemistriesevaluated.

Example 144 Protein Production of Factor VII in HeLa Cells

The day before transfection, 15,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 250 ng ofFactor VII modified RNA (mRNA sequence shown in SEQ ID NO: 1623; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) with the chemical modification described in Table 191, was dilutedin 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island,N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) wasused as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM. After 5 minutes of incubationat room temperature, both solutions were combined and incubated anadditional 15 minute at room temperature. Then the 20 ul combinedsolution was added to the 100 ul cell culture medium containing the HeLacells and incubated at room temperature. A control of untreated cellswas also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing factor VII were collected and centrifuged at 10.000 rcf for 2minutes. The cleared supernatants were then analyzed with a HyphenBiomed Chromogenic kit (Aniara, West Chester Ohio) according to themanufacturer instructions. All samples were diluted until the determinedvalues were within the linear range of the ELISA standard curve. Theamount of protein produced compared to the untreated samples is shown inTable 191 and FIG. 34.

TABLE 191 Protein Production Increase over untreated Sample (pg/ml)5-methylcytosine and pseudouridine (5mC and pU) 1.236 5-methylcytosineand 1-methylpsudouridine (5mC and 1.270 1mpU) 25% of uridine modifiedwith 2-thiouridine and 25% 1.299 of cytosine modified with5-methylcytosine (s2U and 5mC) Pseudouridine (pU) 1.2761-methylpseudouridine (1mpU) 1.224 Untreated 1

Example 145 Protein Production of Chemically Modified Insulin Glarine inHeLa Cell Supernatant

The day before transfection, 15,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul Eagle's minimal essential medium (EMEM) (supplemented with 10%fetal calf serum (FCS) and 1× Glutamax) per well in a 24-well cellculture plate (Corning, Manassas, Va.). The cells were grown at 37° C.in 5% CO₂ atmosphere overnight. Next day, 250 ng of insulin glarginemodified RNA (mRNA sequence shown in SEQ ID NO: 21465; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (5mC/pU), fullymodified with 5-methylcytosine and 1-methylpseudouridine (5mC/lmpU),modified where 25% of the cytosines replaced with 5-methylcytosine and25% of the uridines replaced with 2-thiouridine (s2U/5mC), fullymodified with 1-methylpseudouridine (1 mpU) or fully modified withpseudouridine (pU) were diluted in 10 ul final volume of OPTI-MEM®(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul of lipofectamine 2000 was diluted to 10 ul final volume ofOPTI-MEM®. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature. A control of untreated was also analyzed.

After an 18 hour incubation cell culture supernatants of cellsexpressing insulin glargine were collected by lysing cells withimmuno-precipitation (IP) buffer from Pierce Biotechnology (ThermoScientific, Rockford, Ill.) and centrifuged at 10.000 rcf for 2 minutes.The cleared supernatants were diluted 1:2 (1 ml lysate/2 wells/24 wellplate) or 1:5 (1 ml lysate/5 wells/24 well plate) then analyzed with aninsulin-specific ELISA kit (Mercodia AB, Uppsala, Sweden) according tothe manufacturer instructions. All samples were diluted until thedetermined values were within the linear range of the ELISA standardcurve. The amount of protein produced in both studies is shown in Table192 and FIG. 35. In Table 192, “>” means greater than.

TABLE 192 Protein Production Insulin Glargine Sample (mIU/ml)5-methylcytosine and pseudouridine (5mC/pU) 22.7 5-methylcytosine and1-methylpseudouridine (5mC/1mpU) 85.3 25% of the cytosines replaced with5-methylcytosine 44.2 and 25% of the uridines replaced with2-thiouridine (s2U/5mC) pseudouridine (pU) 42.0 1-methylpseudouridine(1mpU) >105 Untreated 0

Example 146 Protein Production of Tissue Factor (Factor 3) in HeLa Cells

The day before transfection, 15,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 250 ng ofTissue Factor (Factor 3) modified RNA (mRNA sequence shown in SEQ ID NO:21466; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) with the chemical modification described inTable 193, was diluted in 10 ul final volume of OPTI-MEM(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul of lipofectamine 2000 was diluted to 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature. A control of untreated cells was also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing tissue factor were collected and centrifuged at 10.000 rcffor 2 minutes. The cleared supernatants were then analyzed with a HyphenBiomed Chromogenic kit (Aniara, West Chester Ohio) according to themanufacturer instructions. All samples were diluted until the determinedvalues were within the linear range of the ELISA standard curve. Theamount of protein produced compared to the untreated samples is shown inTable 193 and FIG. 36. In Table 193, “>” means greater than.

TABLE 193 Protein Production Tissue Factor Protein Sample (pg/ml)5-methylcytosine and pseudouridine (5mC and pU) >2,500 5-methylcytosineand 1-methylpsudouridine (5mC >2,500 and 1mpU) 25% of uridine modifiedwith 2-thiouridine and 669 25% of cytosine modified with5-methylcytosine (s2U and 5mC) Pseudouridine (pU) >2,5001-methylpseudouridine (1mpU) >2,500 Untreated 3.1

Example 147 Protein Production of Chemically Modified Factor XI in HeLaCells

The day before transfection, 15,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 250 ng ofFactor XI modified RNA (mRNA sequence shown in SEQ ID NO: 1625; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) with the chemical modification described in Table 194, was dilutedin 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island,N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) wasused as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM. After 5 minutes of incubationat room temperature, both solutions were combined and incubated anadditional 15 minute at room temperature. Then the 20 ul combinedsolution was added to the 100 ul cell culture medium containing the HeLacells and incubated at room temperature. A control of untreated cellswas also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing factor XI were collected and centrifuged at 10.000 rcf for 2minutes. The cleared supernatants were then analyzed with a analyzedwith a Factor XI-specific ELISA kit (Innovative Research, Novi, Mich.)according to the manufacturer instructions. All samples were diluteduntil the determined values were within the linear range of the ELISAstandard curve. The amount of protein produced compared to the untreatedsamples is shown in Table 194 and FIG. 37. In Table 194, “>” meansgreater than.

TABLE 194 Protein Production Factor XI Protein Sample (ng/ml)5-methylcytosine and pseudouridine (5mC and pU) >500 5-methylcytosineand 1-methylpsudouridine (5mC and >500 1mpU) 25% of uridine modifiedwith 2-thiouridine and 25% 42 of cytosine modified with 5-methylcytosine(s2U and 5mC) Pseudouridine (pU) >500 1-methylpseudouridine (1mpU) >500Untreated 9

Example 148 Protein Production of Factor XI in HeLa Cell Supernatant

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 250 ng ofFactor XI modified RNA (mRNA sequence shown in SEQ ID NO: 1625; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) fully modified with 5-methylcytosine and 1-methylpseudouridine,was diluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM. After 5 minutes of incubationat room temperature, both solutions were combined and incubated anadditional 15 minute at room temperature. Then the 20 ul combinedsolution was added to the 100 ul cell culture medium containing the HeLacells and incubated at room temperature. A control of erythropoietin(EPO) modified mRNA (mRNA sequence shown in SEQ ID NO: 1638; polyA tailof approximately 160 nucleotides not shown in sequence; cap, Cap1, fullymodified with 5-methylcytosine and pseudouridine) and untreated cellswas also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing plasminogen was collected and centrifuged at 10.000 rcf for 2minutes. The cleared supernatants were then analyzed with a FactorXI-specific ELISA kit (Innovative Research, Novi, Mich.) according tothe manufacturer instructions. All samples were diluted until thedetermined values were within the linear range of the ELISA standardcurve. The amount of protein produced is shown in Table 195 and FIG. 38.

TABLE 195 Protein Production Factor XI Protein Sample (ng/ml) Factor XI105 EPO 0.3 Untreated 1.2

Example 149 Protein Production of Insulin Aspart in HeLa Cells

The day before transfection, 15,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 250 ng ofInsulin Aspart modified RNA (mRNA sequence shown in SEQ ID NO: 21467;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) with the chemical modification described in Table 196, wasdiluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM. After 5 minutes of incubationat room temperature, both solutions were combined and incubated anadditional 15 minute at room temperature. Then the 20 ul combinedsolution was added to the 100 ul cell culture medium containing the HeLacells and incubated at room temperature. A control of untreated cellswas also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing Insulin Apart were collected and centrifuged at 10.000 rcffor 2 minutes. The cleared supernatants were then analyzed with anInsulin-specific ELISA kit (Mercodia AB, Uppsala, Sweden) according tothe manufacturer instructions. All samples were diluted until thedetermined values were within the linear range of the ELISA standardcurve. The amount of protein produced compared to the untreated samplesis shown in Table 196 and FIG. 39. In Table 196, “>” means greater than.

TABLE 196 Protein Production Insulin Aspart Sample (mIU/ml)5-methylcytosine and pseudouridine (5mC and pU) >500 5-methylcytosineand 1-methylpsudouridine (5mC and >500 1mpU) 25% of uridine modifiedwith 2-thiouridine and 25% >500 of cytosine modified with5-methylcytosine (s2U and 5mC) Pseudouridine (pU) >5001-methylpseudouridine (1mpU) >500 Untreated 0

Example 150 Protein Production of Insulin Lispro in HeLa Cells

The day before transfection, 15,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 250 ng ofInsulin Lispro modified RNA (mRNA sequence shown in SEQ ID NO: 21468;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) with the chemical modification described in Table 197, wasdiluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM. After 5 minutes of incubationat room temperature, both solutions were combined and incubated anadditional 15 minute at room temperature. Then the 20 ul combinedsolution was added to the 100 ul cell culture medium containing the HeLacells and incubated at room temperature. A control of untreated cellswas also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing Insulin Lispro were collected and centrifuged at 10.000 rcffor 2 minutes. The cleared supernatants were then analyzed with aninsulin-specific ELISA kit (Mercodia AB, Uppsala, Sweden) according tothe manufacturer instructions. All samples were diluted until thedetermined values were within the linear range of the ELISA standardcurve. The amount of protein produced compared to the untreated samplesis shown in Table 197 and FIG. 40. In Table 197, “>” means greater than.

TABLE 197 Protein Production Insulin Aspart Sample (mIU/ml)5-methylcytosine and pseudouridine (5mC and pU) >500 5-methylcytosineand 1-methylpsudouridine (5mC and >500 1mpU) 25% of uridine modifiedwith 2-thiouridine and 25% >500 of cytosine modified with5-methylcytosine (s2U and 5mC) Pseudouridine (pU) >5001-methylpseudouridine (1mpU) >500 Untreated 0

Example 151 Protein Production of Insulin Glulisine in HeLa Cells

The day before transfection, 15,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 250 ng ofInsulin Glulisine modified RNA (mRNA sequence shown in SEQ ID NO: 21469;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) with the chemical modification described in Table 198, wasdiluted in 10 ul final volume of OPTI-MEM (LifeTechnologies, GrandIsland, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.)was used as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM. After 5 minutes of incubationat room temperature, both solutions were combined and incubated anadditional 15 minute at room temperature. Then the 20 ul combinedsolution was added to the 100 ul cell culture medium containing the HeLacells and incubated at room temperature. A control of untreated cellswas also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing Insulin Lispro were collected and centrifuged at 10.000 rcffor 2 minutes. The cleared supernatants were then analyzed with aninsulin-specific ELISA kit (Mercodia AB, Uppsala, Sweden) according tothe manufacturer instructions. All samples were diluted until thedetermined values were within the linear range of the ELISA standardcurve. The amount of protein produced compared to the untreated samplesis shown in Table 198 and FIG. 41. In Table 198, “>” means greater than.

TABLE 198 Protein Production Insulin Sample (mIU/ml) 5-methylcytosineand pseudouridine (5mC and pU) >500 5-methylcytosine and1-methylpsudouridine (5mC and 1mpU) >500 25% of uridine modified with2-thiouridine and 25% of >500 cytosine modified with 5-methylcytosine(s2U and 5mC) Pseudouridine (pU) >500 1-methylpseudouridine (1mpU) >500Untreated 0

Example 152 Protein Production of Human Growth Hormone in HeLa Cells

The day before transfection, 15,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 250 ng ofhuman growth hormone (hGH) modified RNA (mRNA sequence shown in SEQ IDNO: 1648; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) with the chemical modification described inTable 199, was diluted in 10 ul final volume of OPTI-MEM(LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul of lipofectamine 2000 was diluted to 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20 ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature. A control of untreated cells was also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing human growth hormone were collected and centrifuged at 10.000rcf for 2 minutes. The cleared supernatants were then analyzed with ahuman growth hormone ELISA kit (Cat. No. DGH00; R&D SYSTEMS®,Minneapolis, Minn.) according to the manufacturer instructions. Allsamples were diluted until the determined values were within the linearrange of the ELISA standard curve. The amount of protein producedcompared to the untreated samples is shown in Table 199 and FIG. 42. InTable 199, “>” means greater than.

TABLE 199 Protein Production hGH Sample (pg/ml) 5-methylcytosine andpseudouridine (5mC and pU) >8,000 5-methylcytosine and1-methylpsudouridine (5mC and 1mpU) >8,000 25% of uridine modified with2-thiouridine and 25% of >8,000 cytosine modified with 5-methylcytosine(s2U and 5mC) Pseudouridine (pU) >8,000 1-methylpseudouridine(1mpU) >8,000 Untreated 0

Example 153 Detection of tumor protein 53 Protein: Western Blot

CD1 mice (Harlan Laboratories, South Easton, Mass.) were administeredintravenously lipolexed tumor protein 53 (TP53 or p53) mRNA (mRNAsequence shown in SEQ ID NO: 1670; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (5mC/pU), fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/1mpU), 25% of uridinemodified with 2-thiouridine and 25% of cytosine modified with5-methylcytosine (s2U and 5mC), fully modified with pseudouridine (pU)or fully modified with 1-methylpseudouridine (1 mpU). The mice wereadministered a dose of 2 ug of mRNA complexed with 2 ul Lipofectamine2000 (LifeTechnologies, Grand Island, N.Y.) in 100 ul sterile basal DMEMmedium (w/o additives, LifeTechnologies, Grand Island, N.Y.).

After 6 hours, the animals were sacrificed and serum & spleen are taken.Spleens were transferred to 6-well plates and kept on ice in presence of1 ml PBS. One spleen was cut with a scalpel several times and with arubber cell scraper splenocytes were squeezed out until the PBS turnsturbid due to cell release.

Leaving fibrous components behind, the cells were transferred to a 100um cell strainer (BD Biosciences, San Jose, Calif.) sitting on a 12-wellcell culture plate. By gravity the cells passed through the cellstrainer and were collected beneath in the 12-well culture dish. 1 ml ofPBS was transferred with the free-floating splenocytes to an Eppendorftube and spun for 5 min at 2000 rpm. The PBS was discarded and the cellpellet combined with 500 ul fresh PBS. The spenocytes were resuspendedby brief vortexing for 5 mins at 2000 rpm. The PBS was discarded and 1ml BD Pharmlyse was added to the cell pellet. The splenocytes wereresuspended by brief vortexing. The cells were incubated at roomtemperature for 3 minutes and then spun at 200 rpm for 5 minutes. Thecells were washed twice with 500 ul PBS and spun as described above. Thecells were resuspended with 500 ul of PBS and spun as described.

250 ul of splenocytes were combined with 1× Pharmlyse buffer andvortexed briefly or resuspended with a pipet and then spun for 2 minutesat 2000 rpm.

In one tube, resuspend cell pellet in 500 ul RIPA buffer with proteaseinhibitor cocktail for mammalian cells (BostonBioproducts, Ashland,Mass.) and freeze lysate or continue with BCA assay immediately. In asecond tube, add 250 ul FACS staining kit fixation solution (4%formaldehyde; R and D Systems, Minneapolis, Minn.) and then incubate for10 minutes at room temperature. The cells were washed twice with 500 ulPBS and spun as described above. The cell pellet was resuspended in 500PBS and stored at 4° C.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4×SDS loading buffer (both from Life Technologies, GrandIsland, N.Y.). Samples were heated at 95° C. for 5 min and loaded on thegel. Standard settings were chosen by the manufacturer, 200V, 120 mA andmax. 25W. Run time was 60 min, but no longer than running dye reachingthe lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% BSA in 1× TBS for15 minutes then in 5% BSA in 1× TBS+0.1% Tween for another 15 minutes.Primary antibodies against target proteins were applied in 3 ml of 5%BSA in 1× TBS solution at a 1:500 to 1:2000 dilution for 3 hours at roomtemperature and gentle agitation on an orbital shaker. Membranes arewashed 3 times with 1× TBS/0.1% Tween, 5 minutes each time with gentleagitation. The secondary antibody was conjugated to horse radishperoxidase and binds to the primary antibody antibodies. The secondaryantibody was diluted of 1:1000 to 1:5000 in 5% BSA in 1× TBS andincubated for 3 hrs at RT. Primary and secondary antibodies werepurchased from Abcam (Cambridge, Mass.), Novus Biologicals (Littleton,Colo.), Thermo Fisher (Rockford, Ill.), Millipore (Billerica, Mass.) orR and D systems (Minneapolis, Md.).

At the end of incubation time, the membranes were washed 3 times with1×TBS/0.1% Tween, 5 minutes each time with gentle agitation. Themembranes were developed in 5 ml Pierce WestPico ChemiluminescentSubtrate (Thermo Fisher, Rockford, Ill.) as directed.

As shown with the box in FIGS. 43A and 43B the Western Blot detectedprotein around the expected size of 53 kd for each of the 2 samplesevaluated for each chemistry.

Example 154 Detection of Tuftelin 1 Protein: Western Blot

Human embryonic kidney epithelial (HEK293) were seeded on 96-well plates(Greiner Bio-one GmbH, Frickenhausen, Germany) and plates for HEK293cells were precoated with collagen type 1. HEK293 were seeded at adensity of 35,000 in 100 μl cell culture medium (DMEM, 10% FCS, adding 2mM L-Glutamine, 1 mM Sodiumpyruvate and 1× non-essential amino acids(Biochrom AG, Berlin, Germany) and 1.2 mg/ml Sodiumbicarbonate(Sigma-Aldrich, Munich, Germany)).

Transfected cells were lysed with 20 μl Sample buffer (20% Glycerol, 4%SDS, 100 mM Tris-HCl pH 6.8, 0,2% Bromphenolblue, 5%beta-Mercaptoethanol) per well. Supernatants were precipitated with 4volumes ice-cold acetone and dissolved in sample buffer. Samples wereheated to 95° C. for 5 minutes and run on SDS-polyacrylamide gelscontaining 10% acrylamide.

Proteins were transferred to a nitrocellulose membrane by semidryblotting. Membranes were blocked with 5% skim milk powder in TBS andsubsequently incubated with TUFT1 rabbit polyclonal antibodies (NovusBiologicals, Cat # NBP1-87446) at a dilution of 1:500. Signals weredetected using Donkey anti-rabbit HRP-conjugated secondary antibodies(St. Cruz Biotech, Heidelberg, Germany) and Super Signal West Picodetection regent (Pierce). As shown in FIG. 44 the Western Blot detectedprotein in the 2 ug (lane 2) and 200 ng (lane 3) samples. In FIG. 44,lane 1 is the marker.

Example 155 Detection of Galactokinase 1 Protein: Western Blot

CD1 mice (Harlan Laboratories, South Easton, Mass.) were administeredintravenously lipolexed galactokinase 1 (GALK1) mRNA (mRNA sequenceshown in SEQ ID NO: 21470; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (5mC/pU), fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/lmpU), 25% of uridinemodified with 2-thiouridine and 25% of cytosine modified with5-methylcytosine (s2U and 5mC), fully modified with pseudouridine (pU)or fully modified with 1-methylpseudouridine (1 mpU). The mice wereadministered a dose of 2 ug of mRNA complexed with 2 ul Lipofectamine2000 (LifeTechnologies, Grand Island, N.Y.) in 100 ul sterile basal DMEMmedium (w/o additives, LifeTechnologies, Grand Island, N.Y.).

After 6 hours, the animals were sacrificed and serum & spleen are taken.Spleens were transferred to 6-well plates and kept on ice in presence of1 ml PBS. One spleen was cut with a scalpel several times and with arubber cell scraper splenocytes were squeezed out until the PBS turnsturbid due to cell release.

Leaving fibrous components behind, the cells were transferred to a 100um cell strainer (BD Biosciences, San Jose, Calif.) sitting on a 12-wellcell culture plate. By gravity the cells passed through the cellstrainer and were collected beneath in the 12-well culture dish. 1 ml ofPBS was transferred with the free-floating splenocytes to an Eppendorftube and spun for 5 min at 2000 rpm. The PBS was discarded and the cellpellet combined with 500 ul fresh PBS. The spenocytes were resuspendedby brief vortexing for 5 mins at 2000 rpm. The PBS was discarded and 1ml BD Pharmlyse was added to the cell pellet. The splenocytes wereresuspended by brief vortexing. The cells were incubated at roomtemperature for 3 minutes and then spun at 200 rpm for 5 minutes. Thecells were washed twice with 500 ul PBS and spun as described above. Thecells were resuspended with 500 ul of PBS and spun as described.

250 ul of splenocytes were combined with 1× Pharmlyse buffer andvortexed briefly or resuspended with a pipet and then spun for 2 minutesat 2000 rpm.

In one tube, resuspend cell pellet in 500 ul RIPA buffer with proteaseinhibitor cocktail for mammalian cells (BostonBioproducts, Ashland,Mass.) and freeze lysate or continue with BCA assay immediately. In asecond tube, add 250 ul FACS staining kit fixation solution (4%formaldehyde; R and D Systems, Minneapolis, Minn.) and then incubate for10 minutes at room temperature. The cells were washed twice with 500 ulPBS and spun as described above. The cell pellet was resuspended in 500PBS and stored at 4° C.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4×SDS loading buffer (both from Life Technologies, GrandIsland, N.Y.). Samples were heated at 95° C. for 5 min and loaded on thegel. Standard settings were chosen by the manufacturer, 200V, 120 mA andmax. 25W. Run time was 60 min, but no longer than running dye reachingthe lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% BSA in 1× TBS for15 minutes then in 5% BSA in 1× TBS+0.1% Tween for another 15 minutes.GALK1 rabbit polyclonal antibody (Abcam, Cambridge, Mass.) against GALK1proteins were applied in 3 ml of 5% BSA in 1× TBS solution at a 1:500 to1:2000 dilution for 3 hours at room temperature and gentle agitation onan orbital shaker. Membranes are washed 3 times with 1× TBS/0.1% Tween,5 minutes each time with gentle agitation. The Goat anti-rabbit HRPconjugate (Abcam, Cambridge Mass.) was conjugated to horse radishperoxidase and binds to the GALK1 rabbit polyclonal antibody. Theconjugated antibody was diluted of 1:1000 to 1:5000 in 5% BSA in 1× TBSand incubated for 3 hrs at RT. Primary and secondary antibodies werepurchased from Abcam (Cambridge, Mass.), Novus Biologicals (Littleton,Colo.), Thermo Fisher (Rockford, Ill.), Millipore (Billerica, Mass.) orR and D systems (Minneapolis, Md.).

At the end of incubation time, the membranes were washed 3 times with lxTBS/0.1% Tween, 5 minutes each time with gentle agitation. The membraneswere developed in 5 ml Pierce WestPico Chemiluminescent Subtrate (ThermoFisher, Rockford, Ill.) as directed.

As shown with the box in FIGS. 45A and 45B the Western Blot detectedprotein around the expected size of 30 and 42 kd for each of the 2samples evaluated for each chemistry.

Example 156 Detection of defensin, beta 103A Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO2atmosphere overnight. Next day, 1250 ng of defensing, beta 103A(DEFB103A) mRNA (mRNA sequence shown in SEQ ID NO: 1631; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (5mC/pU) was diluted in250 ul final volume of OPTI-MEM® (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 5.5 ul were diluted in 250 ul final volume ofOPTI-MEM®. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minutes at roomtemperature. Then 500 ul of the combined solution was added to the 3 mlcell culture medium containing the HeLa cells. The plates were thenincubated as described before. A control of untreated cells was alsoevaluated.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitationassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4× SDS loading buffer (both from Life Technologies,Grand Island, N.Y.). Samples were heated at 95° C. for 5 minutes andloaded on the gel. Standard settings were chosen by the manufacturer,200V, 120 mA and maximum 25W. Run time was 60 minutes, but no longerthan running dye reaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. DEFB103A rabbitpolyclonal antibody (Abcam, Cambridge Mass.) against DEFB103A proteinswere applied in 3 ml of 5% BSA in 1× TBS solution at a 1:500 to 1:2000dilution for 3 hours at room temperature and gentle agitation on anorbital shaker. Membranes are washed 3 times with 1× TBS/0.1% Tween, 5minutes each time with gentle agitation. The Donkey anti-rabbit NL557conjugate (R&D Systems, Minneapolis, Minn.) was conjugated to horseradish peroxidase and binds to the DEFB103A rabbit polyclonal antibody.The conjugated antibody was diluted of 1:1000 to 1:5000 in 5% BSA in 1×TBS and incubated for 3 hours at room temperature. As shown with the boxin FIG. 46 the Western Blot detected protein in each of the chemistriesevaluated.

Example 157 Confirmation of Peptide Identity

Proteins can be evaluated using liquid chromatography-mass spectrometryin tandem with mass spectrometry (LC-MS/MS) with quantitativeLC-multiple reaction monitoring (MRM) in order to confirm the identityof the peptide.

The identity of any protein target described herein can be evaluatedusing the liquid chromatography-mass spectrometry in tandem with massspectrometry (LC-MS/MS) with quantitative LC-multiple reactionmonitoring (MRM) Assay (Biognosys AG, Schlieren Switzerland). HeLa celllysates containing protein expressed from modified mRNA are evaluatedusing LC-MS/MS with quantitative LC-MRM Assay (Biognosys, SchlierenSwitzerland) in order to confirm the identity of the peptides in thecell lysates. The identified peptide fragments are compared againstknown proteins including isoforms using methods known and/or describedin the art.

A. Sample Preparation

Protein in each sample in lysis buffer is reduced by incubation for 1hour at 37° C. with 5 mM tris(2-carboxyethyl)phosphine (TCEP).Alkylation is carried out using 10 mM iodoacetamide for 30 minutes inthe dark at room temperature. Proteins are digested to peptides usingtrypsin (sequence grade, PromegaCorporation, Madison, Wis.) at aprotease: protein ratio of 1:50. Digestion is carried out overnight at37° C. (total digestion time is 12 hours). Peptides are cleaned up formass spectrometric analysis using C18 spin columns (The Nest Group,Southborough, Mass.) according to the manufacturer's instructions.Peptides are dried down to complete dryness and resuspended in LCsolvent A (1% acetonitrile, 0.1% formic acid (FA)). All solvents areHPLC-grade from SIGMA-ALDRICH® (St. Louis, Mo.) and all chemicals, wherenot stated otherwise, are obtained from SIGMA-ALDRICH® (St. Louis, Mo.).

B. LC-MS/MS and LC-MRM

Peptides are injected to a packed C18 column (Magic AQ, 3 um particlesize, 200 Å pore size, Michrom Bioresources, Inc (Auburn, Calif.); 11 cmcolumn length, 75 um inner diameter, New Objective (Woburn, Mass.)) on aProxeon Easy nLC nano-liquid chromatography system for all massspectrometric analysis. LC solvents are A: 1% acetonitrile in water with0.1% FA; B: 3% water in acetonitrile with 0.1% FA. The LC gradient forshotgun analysis is 5-35% solvent B in 120 minutes followed by 35-100%solvent B in 2 minutes and 100% solvent B for 8 minutes (total gradientlength is 130 minutes). LC-MS/MS shotgun runs for peptide discovery arecarried out on a Thermo Scientific (Thermo Fisher Scientific)(Billerica, Mass.) Q Exactive mass spectrometer equipped with a standardnano-electrospray source. The LC gradient for LC-MRM is 5-35% solvent Bin 30 minutes followed by 35-100% solvent B in 2 minutes and 100%solvent B for 8 minutes (total gradient length is 40 minutes). TheThermo Scientific (Thermo Fisher Scientific) (Billerica, Mass.) TSQVantage triple quadrupole mass spectrometer is equipped with a standardnano-electrospray source. In unscheduled MRM mode for recalibration itis operated at a dwell time of 20 ms per transition. For relativequantification of the peptides across samples, the TSQ Vantage isoperated in scheduled MRM mode with an acquisition window length of 4minutes. The LC eluent is electrosprayed at 1.9 kV and MRM analysis isperformed using a Q1 peak width of 0.7 Da. Collision energies arecalculated for the TSQ Vantage by a linear regression according to thevendor's specifications.

C. Assay Design, Data Processing and Analysis

For the generation of LC-MRM assays, the 12 most intense fragment ionsfrom LC-MS/MS analysis are measured in scheduled LC-MRM mode and datawere processed using MQUEST® (Cluetec, Karlsruhe, Germany), the scoringpart of mProphet (Reiter et al, mProphet: Automated data processing andstatistical validation for large-scale SRM experiments, Nature Methods,2011 (8), 430-435; the contents of which are herein incorporated byreference). Assays were validated manually, exact fragment intensitiesare determined and iRTs (indexed retention times) are assigned relativeto Biognosys's iRT-peptides (Escher et al. Using iRT, a normalizedretention time for more targeted measurement of peptides, Proteomics,2012 (12), 1111-1121; the contents of which are herein incorporated byreference).

For the relative quantification of the peptides across the sample seriesthe 8 most intense transitions of each assay are measured across thesample series. Data analysis is carried out using SpectroDive™(Biognosys, Schlieren Switzerland). Total peak areas are compared forthe selected peptides and a false discover rate of 0.05 is applied.Peptides with a Qvalue below 0.05 are excluded and considered notdetected in the respective sample.

Example 158 Confirmation of Peptide Identity from Modified mRNAContaining Chemical Modifications

Cell lysates containing protein produced from low density lipoproteinreceptor (LDLR) modified mRNA (mRNA sequence shown in SEQ ID NO: 21463;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and pseudouridine),ornithine carbamoyltransferase (OTC) modified mRNA (mRNA sequence shownin SEQ ID NO: 1659; polyA tail of approximately 160 nucleotides notshown in sequence; 5′ cap, Cap1; fully modified with 5-methylcytosineand pseudouridine), wild-type apolipoprotein A-I (APOA1 wt) modifiedmRNA (mRNA sequence shown in SEQ ID NO: 21453; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine), Hepcidin (HEPC)modified mRNA (mRNA sequence shown in SEQ ID NO: 21471; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and 1-methylpseudouridine),hemochromatosis type 2 (HFE2) modified mRNA (mRNA sequence shown in SEQID NO: 21472; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine and1-methylpseudouridine) or fumarylacetoacetate hydrolase (FAH) modifiedmRNA (mRNA sequence shown in SEQ ID NO: 21473; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and 1-methylpseudouridine) were evaluatedusing the LC-MS/MS with quantitative LC-MRM as described in Example 157.Peptide fragments identified for the evaluated proteins are shown inTable 200. All peptides were specific for parent protein and LDLR andHFE2 was specific for the parent protein and its isoforms. In Table 200,“Uniprot ID” refers to the protein identifier from the UniProt databasewhen the peptide fragment sequences were blasted against all reviewproteins in the database. Housekeeping proteins used to evaluate theprotein in the cell lysates are shown in Table 201.

TABLE 200 Protein and Peptide Fragment Sequences Peptide FragmentPeptide SEQ ID Uniprot Protein Fragment Sequence NO ID APOA1LLDNWDSVTSTFSK 21474 P02647 APOA1 WQEEMELYR 21475 P02647 APOA1 LHELQEK21476 P02647 APOA1 VQPYLDDFQK 21477 P02647 FAH ASSVVVSGTPIR 21478 P16930FAH GEGMSQAATICK 21479 P16930 FAH HLFTGPVLSK 21480 P16930 FAHIGVAIGDQILDLSIIK 21481 P16930 FAH LGEPIPISK 21482 P16930 FAHVFLQNLLSVSQAR 21483 P16930 FAH WEYVPLGPFLGK 21484 P16930 FAHAHEHIFGMVLMNDWSAR 21485 P16930 FAH GTKPIDLGNGQTR 21486 P16930 FAHHQDVFNQPTLNSFMGLGQAAWK 21487 P16930 FAH QHATNVGIMFR 21488 P16930 HEPCASWMPMFQR 21489 P81172 HEPC DTHFPICIFCCGCCHR 21490 P81172 LDLR MICSTQLDR21491 P01130, P01130-4, P01130-3, P01130-2 LDLR LAHPFSLAVFEDK 21492P01130, P01130-4, P01130-3, P01130-2 LDLR NVVALDTEVASNR 21493 P01130,P01130-4, P01130-3, P01130-2 LDLR TCSQDEFR 21494 P01130, P01130-4 OTCFGMHLQAATPK 21495 P00480 OTC KPEEVDDEVFYSPR 21496 P00480 OTC LAEQYAK21497 P00480 OTC LQAFQGYQVTMK 21498 P00480 OTC SLGMIFEK 21499 P00480 OTCVAASDWTFLHCLPR 21500 P00480 OTC GEYLPLLQGK 21501 P00480 OTCGLTLSWIGDGNNILHSIMMSAAK 21502 P00480 OTC LLLTNDPLEAAHGGNVLITDTWIS 21503P00480 MGQEEEK OTC LSTETGFALLGGHPCGLTTQDIHL 21504 P00480 GVNESLT OTCYMLWLSADLK 21505 P00480 HFE2 CNAEYVSSTLSLR 21506 Q6ZVN8 HFE2 GGGVGSGGLCR21507 Q6ZVN8 HFE2 GPALPGAGSGLPAPDPCDYEGR 21508 Q6ZVN8, Q6ZVN8-2 HFE2AFLPDLEK 21509 Q6ZVN8, Q6ZVN8- 2, Q6ZVN8-3 HFE2 LHGRPPGFLHCASFGDPHVR21510 Q6ZVN8, Q6ZVN8-2 HFE2 NMQECIDQK 21511 Q6ZVN8, Q6ZVN8-2

TABLE 201 Housekeeping Proteins Peptide Peptide Fragment Fragment SEQ IDUniprot Protein Sequence NO ID Beta-actin (ACTB) VAPEEHPVLLTEA 21512P60709 PLNPK Glyceraldehyde-3- VVDLMAHMASK 21513 P04406 phosphatedehydrogenase (G3P) Heat shock protein HLEINPDHPIVE 21514 P08238 HSP90-beta TLR (HS90B) Heat shock protein YIDQEELNK 21515 P08238 HSP90-beta (HS90B) L-lactate DQLIYNLLK 21516 P00338 dehydrogenase A chain(LDHA) L-lactate GEMMDLQHGSLF 21517 P00338 dehydrogenase A LR chain(LDHA) Phosphoglycerate ALESPERPFLAIL 21518 P00558 kinase 1 (PGK1) GGAKPhosphoglycerate LGDVYVNDAFGTA 21519 P00558 kinase 1 (PGK1) HR 60Sacidic ribosomal IIQLLDDYPK 21520 P05388 protein P0 (RLA0)

Example 159 Confirmation and of Peptide Identity from ChemicallyModified mRNA

Cell lysates containing protein produced from cytotoxicT-lymphocyte-associated protein 4 (CTLA4) modified mRNA (mRNA sequenceshown in SEQ ID NO: 21521; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1), serpin peptidase inhibitor, GladeA (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1) modifiedmRNA (mRNA sequence shown in SEQ ID NO: 21522; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1),sphingomyelin phosphodiesterase 1 (SMPD1) modified mRNA (mRNA sequenceshown in SEQ ID NO: 21523; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1), hypoxanthine-guaninephosphoribosyltransferase (HPRT1) modified mRNA (mRNA sequence shown inSEQ ID NO: 21524; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1), 4-hydroxyphenylpyruvate dioxigenase (HPD)modified mRNA (mRNA sequence shown in SEQ ID NO: 21525; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1), bonemorphogenic protein-7 (BMP7) modified mRNA (mRNA sequence shown in SEQID NO: 21526; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1), lecithin-cholesterol acyltransferase (LCAT)modified mRNA (mRNA sequence shown in SEQ ID NO: 21527; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1),apoptosis-inducing factor short isoform 1 (AIFsh) modified mRNA (mRNAsequence shown in SEQ ID NO: 21528; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1), tumor protein 53 (TP53or P53) modified mRNA (mRNA sequence shown in SEQ ID NO: 1670; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1), argininosuccinate synthetase (ASS1) modified mRNA (mRNA sequenceshown in SEQ ID NO: 21529; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1), KIT ligand/stem cell factor(KITLG) modified mRNA (mRNA sequence shown in SEQ ID NO: 21530; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1), neuregulin 1, β2a isoform (NRG1) modified mRNA (cDNA sequenceshown in SEQ ID NO: 21531; the T7 promoter, 5′ untranslated region (UTR)and 3′ UTR used in in vitro transcription (IVT) shown in the sequence),vasoactive intestinal peptide (VIP) modified mRNA (mRNA sequence shownin SEQ ID NO: 21532; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1), arylsulfatase B (ARSB) modified mRNA(mRNA sequence shown in SEQ ID NO: 1618; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1),6-pyrovoyltetrahydropterin synthase (PTS) modified mRNA (mRNA sequenceshown in SEQ ID NO: 1609; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1), fibronectin type IIIdomain-containing protein 5 (FNDC5 or Irisin) modified mRNA (cDNAsequence shown in SEQ ID NO: 21533; the T7 promoter, 5′ untranslatedregion (UTR) and 3′ UTR used in in vitro transcription (IVT) shown inthe sequence), amelogenin (AMELY) modified mRNA (mRNA sequence shown inSEQ ID NO: 1613; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1), aldolase A (ALDOA) modified mRNA (mRNAsequence shown in SEQ ID NO: 21534; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1), nerve growth factor(NGF) modified mRNA (mRNA sequence shown in SEQ ID NO: 21535; polyA tailof approximately 140 nucleotides not shown in sequence; 5′ cap, Cap1),glycogen synthase 2 (GYS2) modified mRNA (mRNA sequence shown in SEQ IDNO: 21536; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1), GTP cyclohydrolase 1 (GCH1) modified mRNA (mRNAsequence shown in SEQ ID NO: 1649; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1), hepatocyte growthfactor (HGF) modified mRNA (mRNA sequence shown in SEQ ID NO: 21537;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1), ectodysplasin-A (EDA) modified mRNA (mRNA sequence shown inSEQ ID NO: 1636; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1), arginase (ARG1) modified mRNA (mRNA sequenceshown in SEQ ID NO: 21538; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1), serum amyloid P (APCS) modifiedmRNA (mRNA sequence shown in SEQ ID NO: 21539; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1),phosphorylase kinase, gamma 2 (PHKG2) modified mRNA (mRNA sequence shownin SEQ ID NO: 21540; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1), 685eoxyribonuclease I (DNASE1)modified mRNA (mRNA sequence shown in SEQ ID NO: 1632; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1),Exenatide modified mRNA (mRNA sequence shown in SEQ ID NO: 21541; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1), glycogenin-1 (PYGL) modified mRNA (mRNA sequence shown in SEQ IDNO: 21542; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1), alpha-galactosidase (GLA) modified mRNA (mRNAsequence shown in SEQ ID NO: 1640; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1), alpha-L-iduronidase(IDUA) modified mRNA (mRNA sequence shown in SEQ ID NO: 1652; polyA tailof approximately 140 nucleotides not shown in sequence; 5′ cap, Cap1),galactokinase-1 (GALK1) modified mRNA (mRNA sequence shown in SEQ ID NO:21470; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andpseudouridine (5mC and pU), fully modified with 5-methylcytosine and1-methylpsudouridine (5mC and lmpU), modified where 25% of uridinemodified with 2-thiouridine and 25% of cytosine modified with5-methylcytosine (s2U and 5mC), fully modified with pseudouridine (pU),or fully modified with 1-methylpseudouridine (1 mpU) were evaluatedusing the LC-MS/MS with quantitative LC-MRM as described in Example 157.Peptide fragments identified for the evaluated proteins are shown inTable 202.

TABLE 202 Proteins and Peptide Fragment Sequences Peptide 5mC 5mC s2UFragment and and and SEQ ID NO pU 1mpU 5mC pU 1mpU CTLA4 AMDTGLYICK21543 YES — YES — YES GIASFVCEYASP 21544 — — YES — — GK SERPINA1ITPNLAEFAFSLYR 21545 — YES — YES YES LSITGTYDLK 21546 — YES — — — SMPD1IGGFYALSPYPG 21547 — YES — YES YES HPRT1 DLNHVCVISETG 21548 YES YES YESYES YES EMGGHHIVALC 21549 YES — — YES YES HPD AFEEEQNLR 21550 YES YESYES YES YES EMGDHLVK 21551 — YES — — YES EVVSHVIK 21552 — YES YES — YESBMP7 ATEVHFR 21553 YES YES — YES YES ESDLFLLDSR 21554 — — — — YESIPEGEAVTAAEF 21555 YES YES YES YES YES LCAT LDKPDVVNWMC 21556 — YES — —YES LEPGQQEEYYR 21557 — YES — YES YES SSGLVSNAPGVQ 21558 — YES YES YESYES AIFsh DGEQHEDLNEV 21559 — — — — YES TGGLEIDSDFGG 21560 YES — — YESYES P53 ELNEALELK 21561 — — — — YES SVTCTYSPALNK 21562 — — — YES YESTCPVQLWVDSTP 21563 — — — — YES ASS1 APNTPDILEIEFK 21564 YES YES — — YESFAELVYTGFWHS 21565 YES YES — YES YES FELSCYSLAPQIK 21566 YES YES YES YESYES KITLG AVENIQINEEDN 21567 YES YES — YES YES LFTPEEFFR 21568 YES YES —YES YES LVANLPK 21569 YES YES — — — NRG1 ASLADSGEYMC 21570 YES YES YES —YES VIP HADGVFTSDFSK 21571 YES YES YES YES YES HSDAVFTDNYTR 21572 YESYES YES YES YES ARSB ELIHISDWLPTLV 21573 — YES YES YES YES GVGFVASPLLK21574 — YES YES YES — HSVPVYFPAQDP 21575 — YES YES YES YES PTS FLSDEENLK21576 YES YES YES YES YES VYETDNNIVVYK 21577 YES YES — YES YESYMEEAIMQPLD 21578 YES YES — YES YES FNDC5 SASETSTPEHQG 21579 — YES — YESYES AMELY IALVLTPLK 21580 YES — — YES YES ALDOA ALANSLACQGK 21581 YESYES YES YES YES ALQASALK 21582 YES YES YES YES YES GILAADESTGSIA 21583YES YES YES YES YES NGF LQHSLDTALR 21584 — YES — — YES GYS2 TQVEQCEPVND21585 — — — — YES GCH1 AASAMQFFTK 21586 YES YES YES YES YES IVEIYSR21587 YES YES YES YES YES VHIGYLPNK 21588 YES YES YES YES YES HGFGEEGGPWCFTSN 21589 YES YES — YES YES HIFWEPDASK 21590 — — — — YESNPDGSESPWCFT 21591 — YES — — YES EDA NDLSGGVLNDW 21592 — — YES YES YESARG1 GGVEEGPTVLR 21593 YES YES YES YES YES TIGIIGAPFSK 21594 YES YES YESYES YES YFSMTEVDR 21595 YES YES YES YES YES APCS IVLGQEQDSYGG 21596 YESYES — YES YES QGYFVEAQPK 21597 YES YES — — YES VGEYSLYIGR  2158 YES YES— YES YES PHKG2 IMEVTAER 21599 YES YES YES YES YES LLQVDPEAR 21600 — YES— YES YES LSPEQLEEVR 21601 YES YES YES YES YES DNASE1 IAAFNIQTFGETK21602 YES YES YES YES YES IVVAGMLLR 21603 YES — — YES YES YDIALVQEVR21604 YES YES YES YES YES Exenatide LFIEWLK 21605 YES YES YES — YES PYGLAPNDFNLR 21606 YES YES YES YES YES DYYFALAHTVR 21607 YES YES YES YES YESVLYPNDNFFEGK 21608 YES YES YES YES YES GLA FDGCYCDSLENL 21609 YES YESYES YES YES NFADIDDSWK 21610 YES YES YES YES YES SILDWTSFNQER 21611 YESYES YES YES YES IDUA LDYISLHR 21612 YES YES YES YES YES SPLSWGLLR 21613YES YES YES YES YES THWLLELVTTR 21614 YES YES YES YES YES GALK1GHALLIDCR 21615 YES YES YES YES YES LAVLITNSNVR 21616 YES YES YES YESYES LQFPLPTAQR 21617 YES YES YES YES YES

Example 160 Confirmation of Peptide Identity and Comparison toRecombinant Protein

Liquid chromatography (LC)-mass spectrometry (MS) analysis, LC-MS/MStryptic peptide mapping/sequencing analysis and 2-dimensionalfluorescence differential gel electrophoresis (2-D DIGE) were conductedto confirm the expression of ornithine carbamoyltransferase (OTC)modified mRNA (mRNA sequence shown in SEQ ID NO: 1659; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine or UDPglucuronosyltransferase 1 family, polypeptide Al (UGT1A1) modified mRNA(mRNA sequence shown in SEQ ID NO: 21464; polyA tail of approximately140 nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and 1-methylpseudouridine in HeLa cells transfectedwith either transcript. The following materials were used: HPLC gradewater (EMD Chemicals Inc., Gibbstown, N.J.); formic acid (90%) (J.T.Baker, Phillipsburg, N.J.); iodoacetamide (Sigma Aldrich, St. Louis,Mo.); methanol (EMD Chemicals Inc., Gibbstown, N.J.);Tris(2-carboxyethyl)phosphine hydrochloride solution (TCEP), 0.5 M(Sigma Aldrich, St. Louis, Mo.); Tris(hydroxymethyl)aminomethane buffersubstance (Tris buffer), pH 8.0 (Fluka-Sigma Aldrich, St. Louis, Mo.);calcium chloride dehydrated ACS reagent (CaCl₂) (Sigma Aldrich, St.Louis, Mo.); OTC reference protein [purified recombinant full lengthhuman OTC protein, (Abcam, Cambridge, Mass.)]; UGT1A1 reference protein[purified recombinant human UGT1A1 protein (OriGene Technologies,Rockville, Md.).

Hela cells were transfected with mRNA encoding OTC and UGT1A1. Aftertransfection, cells were lysed and centrifuged to form a cell pellet.The total protein concentration of each sample was determined bybicinchoninic acid assay (BCA assay). The cellular pellet fromuntransfected HeLa cells had a total protein concentration of 1707 μg/mLwhile cellular pellets from OTC and UGT1A1 transfected HeLa cells hadtotal protein concentrations of 3947 μg/mL and 2609 μg/mL, respectively.

For tryptic peptide mapping/sequencing, recombinant proteins as well ascellular pellets were subjected to typsin digestion. Recombinant OTC andUGT1A1 were prepared in 100 mM Tris buffer with 3 mM calcium chloride.The proteins (5 μg each) were reduced with 2 mM TCEP then denatured at100° C. for five minutes. The samples were cooled to room temperaturethen alkylated with 2 mM iodoacetamide. The alkylated samples werefurther reduced with 0.5 mM TCEP. The samples were treated with trypsinovernight at 37° C. in a water bath. The digested samples were removedfrom the water bath and quenched with 2% formic acid in water. Cellpellets were prepared in 100 mM Tris buffer with 3 mM calcium chloride,pH 8.0. The reconstituted cell pellets and supernatant samples (50 μgeach) were reduced with 2 mM TCEP then denatured at 100° C. for fiveminutes. The samples were cooled to room temperature then alkylated with100 mM iodoacetamide. The alkylated samples were further reduced with0.5 mM TCEP. The samples were treated with trypsin overnight at 37° C.in a water bath. The digested samples were removed from the water bathand quenched with 2% formic acid in water.

The tryptic peptides of recombinant proteins, cell pellets, andsupernatant samples were injected onto a 150×2.1 mm Xbridge BEH300 C18column. The column heater was set at 35° C. Mobile Phase A was 0.1%formic acid in water. Mobile Phase B was 0.1% formic acid in 90/10acetonitrile/water (v/v). The mass spectra of the digestion productswere acquired with an ABSciex API QSTAR ELITE quadrupole time-of-flightmass spectrometer (AB Sciex, Foster City, Calif.). Data were acquired intime-of-flight (TOF) MS and MS/MS modes using positive electrosprayionization (ESI). The Turbo IonSpray interface was set at 500° C. andmaintained at a turbospray voltage of 5.0 kV with a declusteringpotential of 35 V. Ionization was assisted with a nebulizer and ionspraygas (nitrogen) set at 40 and 35 (arbitrary units) respectively. The datawere acquired and processed using Analyst QS 2.0 software (AB Sciex,Foster City, Calif.).

The LC-MS data were compared against the predicted/theoretical trypticdigestion products of OTC and UGT1A1 proteins using BioAnalyst software(AB Sciex, Foster City, Calif.). The digestion products of therecombinant proteins were used as reference peptides for peptidematching in the digested cell pellets and supernatant. The LC-MS andLC-MS/MS data were also mapped and sequenced to specific trypticdigestion products of the HeLa cell derived proteins. The peptidesequence of tryptic peptides were confirmed based on the LC-MS/MSproduct ion spectra.

OTC protein was present in the cell pellet of the HeLa cells transfectedwith the OTC mRNA. Peptide mapping identified sixteen OTC peptides inthe tryptic digest of the cell pellet. The product ion spectra of thecell derived peptides matched well with those of the recombinantprotein. A database search of human proteins indicated the sequences ofthe thirteen unmodified peptides were specific only to human OTC. UGT1A1protein was expressed by the HeLa cells transfected with the UGT1A1mRNA. Seven UGT1A1 peptides were identified in the tryptic digest of thecell pellet based on exact mass assignment and retention time match tothe recombinant UGT1A1 protein. The identities of the matched peptideswere confirmed by LC-MS/MS. Based on a search of human proteins, four ofthe seven observed tryptic peptides was specific to human UGT1A proteinswhile one of the seven observed tryptic peptides was specific only forUGT1A1. Table 203 shows the peptides identified from the HeLa celllysates for UGT1A1 modified mRNA and Table 204 shows the peptidesidentified from OTC modified mRNA.

TABLE 203 UGT1A1 Peptide Fragments Peptide Fragment Sequence SEQ ID NOAMAIADALGKIPQTVLWR 21618 WLPQNDLLGHPMTR 21619 IPQTVLWR 21620 TYPVPFQR21621 AMAIADALGK 21622 ENIMR 21623 SDFVK 21624

TABLE 204 OTC Peptide Fragments Peptide Fragment Sequence SEQ ID NOQKGEYLPLLQGK 21625 VLSSMADAVLAR 21626 FGMHLQAATPK 21627 SLVFPEAENR 21628GEYLPLLQGK 21629 GYEPDASVTK 21630 ILLNNAAFR 21631 QSDLDTLAK 21632NFTGEEIK 21633 SLGMIFEK 21634 GRDLLTLK 21635 LAEQYAK 21636 DLLTLK 21637

The protein profiles of the cell pellet samples were furthercharacterized by two-dimensional fluorescence differential gelelectrophoresis. The test samples were treated with differentfluorescent dyes. The contents of the samples were resolved usingisoelectric focusing (IEF) in the first dimension and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the seconddimension. The protein profiles of the OTC and UGT1A1 cell pellets werecompared. DeCycler Differential Analysis Software (GE Healthcare,Buckinghamshire, UK) was used for data analysis. The presence of OTC andUGT1A1 in the HeLa cells was confirmed by the results of the analysis.

Example 161 Confirmation of Peptide Identity from Modified mRNAContaining Chemical Modifications

Cell lysates containing protein produced from cathelicidin antimicrobialpeptide (CAMP) modified mRNA (mRNA sequence shown in SEQ ID NO: 1621;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1), CAP18 modified mRNA (mRNA sequence shown in SEQ ID NO:21638; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1), ciliary neurotrophic factor (CNTF) modifiedmRNA (mRNA sequence shown in SEQ ID NO: 21639; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1),follicle stimulating hormone, beta polypeptide (FSHB) modified mRNA(mRNA sequence shown in SEQ ID NO: 21640; polyA tail of approximately140 nucleotides not shown in sequence; 5′ cap, Cap1), interferon, alpha2 (IFNA2) modified mRNA (mRNA sequence shown in SEQ ID NO: 1654; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1), interferon, beta 1, fibroblast (IFNB1) modified mRNA (mRNAsequence shown in SEQ ID NO: 1655; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1), methylmalonyl-CoAmutase, mitochondrial (MUTA) modified mRNA (mRNA sequence shown in SEQID NO: 1658; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1), galectin 3 (LEG3) modified mRNA (mRNA sequenceshown in SEQ ID NO: 21641; poly A tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1), transforming growth factor beta-3(TGFB3) modified mRNA (mRNA sequence shown in SEQ ID NO: 21642; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1), or tuftelin (TUFT1) modified mRNA (mRNA sequence shown in SEQ IDNO: 1669; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andpseudouridine (5mC and pU), fully modified with 5-methylcytosine and1-methylpsudouridine (5mC and lmpU), modified where 25% of uridinemodified with 2-thiouridine and 25% of cytosine modified with5-methylcytosine (s2U and 5mC), fully modified with pseudouridine (pU),or fully modified with 1-methylpseudouridine (1 mpU) were evaluatedusing the LC-MS/MS with quantitative LC-MRM as described in Example 157.Peptide fragments identified for the evaluated proteins are shown inTable 205. In Table 205, “Uniprot ID” refers to the protein identifierfrom the UniProt database when the peptide fragment sequences wereblasted against all review proteins in the database.

TABLE 205 Protein and Peptide Fragment Sequences Peptide Fragment 5mC5mC s2U SEQ and and and Uniprot ID NO pU 1mpU 5mC pU 1mpU ID CAMPCMGTVTLNQAR 21643 — Yes Yes Yes Yes P49913 FALLGDFFR 21644 — Yes Yes YesYes P49913 TTQQSPEDCDFK 21645 Yes Yes Yes Yes Yes P49913 CAP18CVGTVILNQAR 21646 Yes Yes Yes Yes Yes NA SSDANLYR 21647 Yes Yes Yes YesYes P49913 CNTF LQENLQAYR 21648 Yes Yes Yes Yes Yes P26441 TFHVLLAR21649 Yes Yes Yes Yes Yes P26441 VLQELSQWTV 21650 Yes Yes Yes Yes YesP26441 FSHB CDSDSTDCTVR 21651 Yes Yes Yes Yes Yes P01225 ELVYETVR 21652— — Yes — Yes P01225 VPGCAHHADSL 21653 Yes Yes Yes Yes Yes P01225YTYPVATQCHC IFNA2 SFSLSTNLQESL 21654 — Yes — Yes Yes P01563 TLMLLAQMR21655 — Yes — Yes Yes P01563 YSPCAWEVVR 21656 — — — — Yes P01563 IFNB1LLWQLNGR 21657 — Yes — Yes Yes P01574 LMSSLHLK 21658 — — — Yes YesP01574 LEG3 GNDVAFHFNPR 21659 Yes Yes Yes Yes Yes P17931 IQVLVEPDHFK21660 Yes Yes Yes Yes Yes P17931 MLITILGTVKPN 21661 Yes Yes Yes Yes YesP17931 MUTA LINEIEEMGGM 21662 Yes Yes Yes Yes Yes P22033 NTQIIIQEESGIP21663 Yes Yes Yes Yes Yes P22033 TGLQAGLTIDE 21664 Yes Yes Yes Yes YesP22033 TGFB3 ALDTNYCFR 21665 Yes Yes Yes Yes Yes P10600 GYYANFCSGPC21666 Yes Yes — Yes Yes P10600 VEQLSNMVVK 21667 Yes Yes Yes Yes YesP10600 TUFT1 EAEQSNVALQR 21668 Yes Yes Yes Yes Yes Q9NNX1 IAYLEAENLEM21669 — Yes Yes Yes Yes Q9NNX1 SEVQYIQEAR 21670 Yes Yes Yes Yes YesQ9NNX1

Example 162 Detection of Low Density Lipoprotein Receptor Expression inCell Culture

A. HeLa cell transfection

HeLa cells were plated into 24-well dishes (Corning Life Sciences,Tewksbury, Mass.) (7.5×10⁴ cells/well) in Eagles Minimal EssentialMedium (EMEM, Life Technologies, Grand Island, N.Y.) supplemented with10% fetal calf serum (FCS, Life Technologies, Grand Island, N.Y.) and 1×glutamax reagent (Life Technologies, Grand Island, N.Y.) culturedovernight under standard cell culture conditions. Transfection solutionswere prepared for each well to be treated by combining 250 ng of lowdensity lipoprotein receptor (LDLR) modified mRNA (mRNA sequence shownin SEQ ID NO: 21463; polyA tail of approximately 140 nucleotides notshown in sequence; 5′ cap, Cap1) fully modified with 5-methylcytosineand pseudouridine, mCherry modified mRNA (mRNA sequence shown in SEQ IDNO: 21439; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) or luciferase modified mRNA (mRNA sequence shown in SEQID NO: 21446; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine) with 50 μl Opti-MEM reagent (Life Technologies, GrandIsland, N.Y.) in a first tube and 1 μl of L2000 transfection reagent(Life Technologies, Grand Island, N.Y.) in 50 μl of Opti-MEM in a secondtube. After preparation, first and second tubes were incubated at roomtemperature for 5 minutes before combining the contents of each.Combined transfection solutions were incubated for 15 minutes at roomtemperature. 100 μl of transfection solution was then added to eachwell. Cells were cultured for an additional 16 hours before continuedanalysis.

B. LDLR Detection by Flow Cytometry

After transfection, medium were removed from cells and 60 μl of 0.25%trypsin (Life Technologies, Grand Island, N.Y.) was added to each well.Cells were trypsinized for 2 minutes before the addition of 24 μl/wellof trypsin inhibitor (Life Technologies, Grand Island, N.Y.). Resultingcell solutions were transferred to 96 well plates (Corning LifeSciences, Tewksbury, Mass.), cells were pelleted by centrifugation (800×gravity for 5 minutes) and supernatants were discarded. Cell pelletswere washed with PBS and resuspended in Foxp3 Fixation/Permeabilizationsolution (eBioscience, San Diego, Calif.) for 45 minutes. Cells werepelleted again by centrifugation (800× gravity for 5 minutes) andresuspended in permeabilization buffer (eBiosciences, San Diego, Calif.)for 10 minutes. Cells were pelleted again by centrifugation (800×gravity for 5 minutes) and washed in permeabilization buffer. Cells werethen treated with primary antibodies directed toward LDLR, followed byphycoerythrin-labeled secondary antibodies. Labeled cells were thencombined with FACS buffer (PBS with 1% bovine serum albumin and 0.1%sodium azide) and transferred to cluster tubes. As shown in FIG. 47,labeled cells were then analyzed by flow cytometry using a BD Accuri (BDBiosciences, San Jose, Calif.).

C. LDLR Detection by Immunofluorescence

Transfected cells were washed with PBS, and treated with fixationsolution (PBS with 4% formaldehyde) for 20 minutes at room temperature.Cells were then washed with PBS and treated withpermeabilization/blocking solution (Tris buffered saline with 5% bovineserum albumin with 0.1% Tween-20). Cells were incubated for 2 hours atroom temperature with gentle agitation before washing 3 times with PBScontaining 0.05% Tween-20. Cells were then treated with or withoutprimary antibodies (goat anti-LDLR, R&D Systems, Minneapolis, Minn.) ornormal IgG controls for 2 hours at room temperature, washed 3 times withPBS containing 0.05% Tween-20 and treated with secondary antibodysolutions containing a 1:200 dilution of donkey anti-goat IgG withfluorescent label (R&D Systems, Minneapolis, Minn.). Cells were againwashed with PBS containing 0.05% Tween-20 and examined by fluorescencemicroscopy imaging. Cells transiently expressing luciferase or mCherrywere examined by fluorescence microscopy without fluorescentimmunostaining

Example 163 Detection of Defensin, Beta 103A by Immunofluorescence

A. HeLa Cell Transfection

HeLa cells are plated into 24-well dishes (Corning Life Sciences,Tewksbury,

MA) (7.5×10⁴ cells/well) in Eagles Minimal Essential Medium (EMEM, Life

Technologies, Grand Island, N.Y.) supplemented with 10% fetal calf serum(FCS, Life Technologies, Grand Island, N.Y.) and 1× glutamax reagent(Life Technologies, Grand Island, N.Y.) cultured overnight understandard cell culture conditions. Transfection solutions are preparedfor each well to be treated by combining 250 ng defensin, beta 103A(DEFB103A) modified mRNA (mRNA sequence shown in SEQ ID NO: 1631; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) or mCherry modified mRNA (mRNA sequence shown in SEQ ID NO: 21439;polyA tail of approximately 160 nucleotides not shown in sequence; 5′cap, Cap1) with 50 μl Opti-MEM reagent (Life Technologies, Grand Island,N.Y.) in a first tube and 1 μl of L2000 transfection reagent (LifeTechnologies, Grand Island, N.Y.) in 50 μl of Opti-MEM in a second tube.After preparation, first and second tubes are incubated at roomtemperature for 5 minutes before combining the contents of each.Combined transfection solutions are incubated for 15 minutes at roomtemperature. 100 μl of transfection solution is then added to each well.Cells are cultured for an additional 16 hours before continued analysis.

B. DEFB103A Detection by Immunofluorescence

Transfected cells are washed with PBS, and treated with fixationsolution (PBS with 4% formaldehyde) for 20 minutes at room temperature.Cells are then washed with PBS and treated withpermeabilization/blocking solution (Tris buffered saline with 5% bovineserum albumin with 0.1% Tween-20). Cells are incubated for 2 hours atroom temperature with gentle agitation before washing 3 times with PBScontaining 0.05% Tween-20. Cells are then left untreated with primaryantibody, treated with anti-DEFB103A primary antibody (Rabbitanti-DEFB103A, Abcam, Cambridge, Mass.) or treated with normal IgGcontrol antibody for 2 hours at room temperature, washed 3 times withPBS containing 0.05% Tween-20 and treated with secondary antibodysolutions containing a 1:200 dilution of donkey anti-rabbit IgG with redfluorescent label (R&D Systems, Minneapolis, Minn.). Cells are againwashed with PBS containing 0.05% Tween-20 and examined by fluorescencemicroscopy imaging. Cells transfected with modified mRNA encodingmCherry are visualized without immunofluorescent staining

Example 164 Confirmation of Protein Expression

Modified mRNA fully modified with 5-methylcytosine and pseudouridine(5mC/pU), fully modified with 5-methylcytosine and 1-methylpseudouridine(5mC/lmpU), modified where 25% of the cytosines replaced with5-methylcytosine and 25% of the uridines replaced with 2-thiouridine(s2U/5mC), fully modified with 1-methylpseudouridine (1 mpU) or fullymodified with pseudouridine (pU) encoding the proteins described inTable 205 were analyzed by fluorescence and/or western blot in order toconfirm the expression of protein and the length of the proteinexpressed. Table 205 describes the sequence of the modified mRNA (polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) or the cDNA (the T7 promoter, 5′ untranslated region (UTR) and 3′UTR used in in vitro transcription (IVT) shown in the sequence) thechemical modification evaluated and the length (in base pairs), ifknown.

TABLE 205 Protein and Expression Modified mRNA SEQ ID 5mC/ 5mC/ s2U/Protein NO pU 1mpU 5mC pU 1mpU Luciferase 21446 Yes Yes Yes Yes YesTumor protein 53 1670 Yes Yes — — Yes (P53 or TP53) 1468 bp 1468 bp 1468bp Phenylalanine 1660 Yes Yes Yes — Yes hydroxylase 1645 bp 1645 bp 16451645 bp (PAH) bp Factor VII (F7) 1623 Yes Yes Yes — Yes 1687 bP 1687 bP1687 1687 bP bP Defensin, beta 1631 Yes Yes Yes Yes Yes 103A 490 bp 490bp 490 490 490 bp (DEFB103A) bp bp 6- 1609 Yes Yes Yes Yes Yespyruvoyltetra- 724 bp 724 bp 724 724 724 bp hydropterin bp bpsynthase(PTS) Serpina 1 21522 Yes Yes — — Yes 1543 bp 1543 bp 1543 bpSphingomyelin 21523 Yes Yes — — Yes phosphodiesterase 2176 bp 2176 bp2176 bp 1, acid lysosomal (SMPD1) Herceptin Light 21452 Yes Yes Yes YesYes Chain 1042 bp 1042 bp 1042 1042 1042 bp bp bp Herceptin Heavy 21451Yes Yes Yes Yes Yes Chain 1753 bp 1753 bp 1753 1753 1753 bp bp bp Glialcell derived 21671 Yes Yes Yes — Yes neurotrophic 922 bp 922 bp 922 922bp factor (GDNF) bp Rhodopsin (RHO) 21672 Yes Yes — — Yes 985 bp 985 bp985 bp Tissue 1661 Yes Yes Yes — Yes Plasminogen 1975 bp 1975 1975 1975bp Activator (PLAT) bp bp Argininosuccinate 21529 Yes Yes — — Yessynthase 1 1525 bp 1525 1525 bp (ASS1) bp UDP 21464 Yes Yes — — Yesglucuronosyl- 1888 bp 1888 1888 bp transferase 1 bp family, polypeptideA1 (UGT1A1) Sirtuin 6 (SIRT6) 1665 Yes Yes Yes — Yes 1354 bp 1354 bp1354 1354 bp bp Thrombopoietin 1667 Yes Yes Yes — Yes (THPO) 3088 bp3088 bp 3088 3088 bp bp Hypoxanthine 21524 Yes Yes — — Yesphosphoribosyl- 943 bp 943 bp 943 bp transferase 1 (HPRT1)Thrombomodulin 21673 Yes Yes — — Yes (THBD) 2014 bp 2014 bp 2014 bpFactor XII (F12) 21674 Yes Yes Yes — Yes 2134 bp 2134 bp 2134 2134 bp bpRelaxin 2 (RLN2) 21675 Yes Yes Yes Yes Yes 844 bp 844 bp 844 844 844 bpbp bp Vasoactive 21532 Yes Yes Yes Yes Yes Intestinal Peptide 799 bp 799bp 799 799 799 bp (VIP) bp bp Lecithin- 21527 Yes Yes Yes — Yescholesterol 1609 bp 1609 bp 1609 1609 bp acyltransferase bp (LCAT)Fibroblast growth 21676 Yes Yes Yes — Yes factor 21 916 bp 916 bp 916916 bp bp OMOMYC 21677 Yes Yes — — Yes 1623 bp 1623 bp 1623 bpApoptosis- 21528 Yes Yes — — Yes inducing factor 1089 bp 1089 bp 1089 bppro-apoptotic isoform (AIFsh) Erythropoietin 1638 Yes — — — — (EPO) 868bp Human Growth 1648 Yes Yes Yes Yes Yes Hormone (hGH) 940 bp 940 bp 940940 940 bp bp bp Glactosidase, 1640 — Yes Yes Yes — alpha (GLA) 1576 bp1576 1576 bp bp Arylsulfatase B 1618 — Yes Yes Yes — 1888 bp 1888 1888bp bp Iduronidase, 1652 — Yes Yes Yes — alpha-L-(IDUA) 2248 bp 2248 2248bp bp interferon, beta 1, 1655 Yes — — — — fibroblast (IFNB) 850 bpCTLA4-Ig 21521 — Yes Yes Yes — 958 bp 958 958 bp bp Interferon alpha 21654 Yes — — — — (IFNA2) 853 bp Factor IX (F9) 1622 Yes Yes Yes — Yes1672 bp 1672 bp 1672 1672 bp bp APOA1 Milano 21455 Yes Yes Yes Yes Yes1090 bp 1090 bp 1090 1090 1090 bp bp bp Factor XI (F11) 1625 Yes Yes Yes— Yes 2164 bp 2164 bp 2164 2164 bp bp CAP18 21638 Yes — — — — 796 bpAquaporin 5 1617 Yes Yes Yes Yes Yes 1084 bp 1084 bp 1084 1084 1084 bpbp bp APOA1 Paris 21454 Yes Yes Yes Yes Yes 1090 bp 1090 bp 1090 10901090 bp bp bp APOA1 21453 Yes Yes Yes Yes Yes 1090 bp 1090 bp 1090 10901090 bp bp bp follicle 21640 Yes — — — — stimulating 673 bp hormone beta(FSH beta) Prothrombin 21462 Yes Yes Yes Yes Yes 2155 bp 2155 bp 21552155 2155 bp bp bp Tissue Factor 21466 Yes Yes Yes Yes Yes (Factor 3)1174 bp 1174 bp 1174 1174 1174 bp bp bp galactokinase 1 21470 — Yes —Yes Yes (GALK1) 1465 bp 1465 1465 bp bp 4- 21525 — — Yes Yes Yeshydroxyphenyl- 1468 1468 1468 bp pyruvate bp bp dioxigenase (HPD) bone21526 — Yes Yes Yes — morphogenic 1582 bp 1582 1582 protein-7 (BMP7) bpbp KIT ligand/stem 21530 — Yes Yes Yes — cell factor 1108 bp 1108 1108(KITLG) bp bp Insulin Glulisine 21469 Yes Yes Yes Yes Yes 619 bp 619 bp619 619 619 bp bp bp Insulin Lispro 21468 Yes Yes Yes Yes Yes 619 bp 619bp 619 619 619 bp bp bp Insulin Aspart 21467 Yes Yes Yes Yes Yes 619 bp619 bp 619 619 619 bp bp bp Insulin Glargine 21465 Yes Yes Yes Yes Yes619 bp 619 bp 619 619 619 bp bp bp LL-37 1621 Yes — — — — 796 bpMethylmalonyl- 1658 Yes — — — — CoA mutase, 2539 bp mitochondrial (MUTA)Ceruloplasmin 1620 Yes — — — — (CP) 3481 bp cDNA SEQ ID 5mC/ 5mC/ s2U/Protein NO pU 1mpU 5mC pU 1mpU Irisin 21532 Yes Yes Yes — Yes 925 bp 925bp 925 925 bp bp

Example 165 Vascular Endothelial Growth Factor Expression and CellVitality

The day before transfection, 100,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 24-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 62 ng, 250ng, 750 ng or 1500 ng of vascular endothelial growth factor (VEGF)modified RNA (mRNA sequence shown in SEQ ID NO: 1672; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine (5mC/pU), fullymodified with 5-methylcytosine and 1-methylpseudouridine (5mC/lmpU),fully modified with 1-methylpseudouridine (1 mpU) or containing nomodifications (unmodified) was mixed with 2 ul Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) as transfection aid and added tothe cells in duplicate.

A. Vascular Endothelial Growth Factor Protein Expression

After an incubation of 24 hours cell culture supernatants of cellsexpressing

VEGF were collected and centrifuged at 10.000 rcf for 2 minutes. Thecleared supernatants were then analyzed with a VEGF-specific ELISA kit(R & D Systems, Minneapolis, Minn.) according to the manufacturerinstructions. All samples were diluted until the determined values werewithin the linear range of the ELISA standard curve. A control ofuntreated cells, cells treated in duplicate with 62 ng or 750 ng ofgreen fluorescent protein (GFP) modified mRNA (mRNA sequence shown inSEQ ID NO: 1672; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andpseudouridine (5mC/pU), fully modified with 5-methylcytosine and1-methylpseudouridine (5mC/lmpU), fully modified with1-methylpseudouridine (1 mpU) or containing no modifications(unmodified) and a control of 250 ng of mCherry modified mRNA (mRNAsequence shown in SEQ ID NO: 21439; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) was also analyzed. Theamount of VEGF produced in pg/ml is shown in Table 206 and in FIG. 48.In FIG. 48, “eGFP” refers to the green fluorescent protein modifiedmRNA, “MC” refers to the mCherry modified mRNA, “UNT-1” refers to theuntreated cells, “G0” means the modified mRNA contains no modifications,“G1” means fully modified with 5-methylcytosine and pseudouridine, “G1”means fully modified with 5-methylcytosine and 1-methyl pseudouridineand “G5” means fully modified with 1-methylpseudouridine.

Cells transfected with VEGF modified mRNA fully modified with5-methylcytosine and 1-methylpseduoruidine or fully modified with1-methylpseudouridine showed the highest expression of VEGF.

TABLE 206 VEGF Protein Production (pg/ml) 5mC/pU 5mC/1mpU 1mpUUnmodified VEGF 62 ng 220500 327409 325500 156545 250 ng 322636 283500168955 45818 750 ng 1264773 2486591 3085091 107864 1500 ng 22317273874500 3465955 238636 GFP 62 ng NA 0 3818 955 750 ng NA 11455 1909 6682Untreated 4773 mCherry 0

B. Vascular Endothelial Growth Factor

After an incubation of 24 hours the medium was removed and cells lysedwith 200 ul per well of the Cell Titer Glo (CTG) assay reagent (PromegaCorporation, Madison, Wis.). After 15 minutes of incubation at roomtemperature, two sets of 10 ul of each lysate were transferred intowhite opaque polystyrene 96-well plates (Corning, Tewksbury, Mass.).Again 100 ul of Cell Titer Glo assay reagent was added to each aliquotof cell lysate. The total volume of 110 ul reagent mix was analyzed on aBioTek Synergy H1 plate reader with a general luminescence program. Themean of duplicate measures were plotted in a graph with untreated HeLacells as reference (1.0=100% vitality). A control of untreated cells induplicate and cells treated with 250 ng of mCherry modified mRNA (mRNAsequence shown in SEQ ID NO: 1672; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) was also evaluated. Thenormalized ATP abundance/cell viability is shown in Table 207 and FIG.49. In FIG. 49, “MC”refers to the mCherry modified mRNA, “UNT-1” and“UNT-2” refers to the untreated cells, “G0” means the modified mRNAcontains no modifications, “G1” means fully modified with5-methylcytosine and pseudouridine, “G1” means fully modified with5-methylcytosine and 1-methyl pseudouridine, “G5” means fully modifiedwith 1-methylpseudouridine and “*” means only one data point wasobtained.

Decreased luminescence activity, which is indicative of loss of cellvitality, was seen in cells transfected with VEGF modified mRNA fullymodified with 5-methylcytosine and pseudouridine or unmodified. Therewas no decrease in cell vitality for cells transfected with VEGFmodified mRNA fully modified with 5-methylcyotosine and1-methylpsuedouridine or fully modified with 1-methylpseudouridine.

TABLE 207 Normalized ATP abundance/Cell Vitality 5mC/pU 5mC/1mpU 1mpUUnmodified 62 ng 1.12 1.19 1.20 1.59 250 ng 0.80 1.11 1.14 0.86 750 ng0.64 1.01 1.04 0.38 1500 ng 0.48 0.99 0.98 0.48 Untreated 0.94 mCherry1.04

Example 166 Protein Production of Erythropoietin in HeLa CellSupernatant

The day before transfection, 100,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 250 ng oferythropoietin (EPO) modified RNA (mRNA sequence shown in SEQ ID NO:1638; polyA tail of approximately 160 nucleotides not shown in sequence;5′ cap, Cap1) fully modified with 5-methylcytosine and pseudouridine(5mC/pU), fully modified with 5-methylcytosine and 1-methylpseudouridine(5mC/lmpU), modified where 25% of uridine modified with 2-thiouridineand 25% of cytosine modified with 5-methylcytosine (s2U and 5mC), fullymodified with pseudouridine (pU), or fully modified with1-methylpseudouridine (1 mpU), was diluted in 10 ul final volume ofOPTI-MEM (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 0.2 ul of lipofectamine 2000 was diluted to 10 ul final volume ofOPTI-MEM. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minute at roomtemperature. Then the 20ul combined solution was added to the 100 ulcell culture medium containing the HeLa cells and incubated at roomtemperature. A control of lipofecatmine 2000 treated cells (L2000) anduntreated cells was also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing EPO was collected and centrifuged at 10.000 rcf for 2minutes. The cleared supernatants were then analyzed with an EPO ELISAkit (Stem Cell Technologies, Vancouver Canada) according to themanufacturer instructions. All samples were diluted until the determinedvalues were within the linear range of the ELISA standard curve. Theamount of protein produced is shown in Table 208.

TABLE 208 Protein Production EPO Sample (pg/ml) 5-methylcytosine andpseudouridine (5mC and pU) 17,900 5-methylcytosine and1-methylpsudouridine (5mC and 81,146 1mpU) 25% of uridine modified with2-thiouridine and 25% of 224,195 cytosine modified with 5-methylcytosine(s2U and 5mC) Pseudouridine (pU) 226,283 1-methylpseudouridine (1mpU)522,375 L2000 597 Untreated 0

Example 167 In vitro expression of Insulin Aspart modified mRNA

The cells were transfected with Insulin Aspart modified mRNA (mRNAsequence shown in SEQ ID NO: 21468; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (5mC/pU), fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/lmpU), modified where25% of uridine modified with 2-thiouridine and 25% of cytosine modifiedwith 5-methylcytosine (s2U and 5mC), fully modified with pseudouridine(pU), or fully modified with 1-methylpseudouridine (1 mpU) which hadbeen complexed with Lipofectamine-2000 from Invitrogen (Carlsbad,Calif.) at the concentration shown in Table 209. The total volume oftransfection mix per 96-well was 50 μl, split in two aliquots 25 μleach. One 25 μl aliquot contained 1 μl of respective mmRNA and 24 μlOpti-MEM, the other 25 μl aliquot contained 0.4 μl Lipofectamine-2000and 24.6 μl Opti-MEM. Where needed, the aliquot containing modified mRNAand Opti-MEM was serially diluted in Opti-MEM. Both aliquots wereincubated separately for 15 min at room temperature. Subsequently, bothaliquots were mixed together and incubated for another 20 min at roomtemperature before finally transferring the total 50 μl transfection mixto a 96-well containing 100 μl cell culture medium plus 35000 HEK293cells. Cells were then incubated for 24 h in a humidified 37° C./5% CO₂cell culture incubator before harvesting the cell culture supernatant orpreparing cell lysate. The protein expression was detected by ELISA andthe protein (pg/ml) is shown in FIG. 50 and in Table 209. In Table 209,“>” means greater than.

TABLE 209 Protein Expression Amount 2 ug 400 ng 80 ng 16 ng 3.2 ng 640pg 128 26 Transfected pg pg Protein >100 >100 >100 103.0 11.4 1.3 0 0(mU/I)

Example 168 In Vitro Expression of Insulin Glarine Modified mRNA

The cells were transfected with Insulin Glargine modified mRNA (mRNAsequence shown in SEQ ID NO: 21465; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (5mC/pU), fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/lmpU), modified where25% of uridine modified with 2-thiouridine and 25% of cytosine modifiedwith 5-methylcytosine (s2U and 5mC), fully modified with pseudouridine(pU), or fully modified with 1-methylpseudouridine (1 mpU) which hadbeen complexed with Lipofectamine-2000 from Invitrogen (Carlsbad,Calif.) at the concentration shown in Table 210. The total volume oftransfection mix per 96-well was 50 μl, split in two aliquots 25 μleach. One 25 μl aliquot contained 1 μl of respective mmRNA and 24 μlOpti-MEM, the other 25 μl aliquot contained 0.4 μl Lipofectamine-2000and 24.6 μl Opti-MEM. Where needed, the aliquot containing modified mRNAand Opti-MEM was serially diluted in Opti-MEM. Both aliquots wereincubated separately for 15 min at room temperature. Subsequently, bothaliquots were mixed together and incubated for another 20 min at roomtemperature before finally transferring the total 50 μl transfection mixto a 96-well containing 100 μl cell culture medium plus 35000 HEK293cells. Cells were then incubated for 24 h in a humidified 37° C./5% CO₂cell culture incubator before harvesting the cell culture supernatant orpreparing cell lysate. The protein expression was detected by ELISA andthe protein (pg/ml) is shown in FIG. 51 and in Table 210.

TABLE 210 Protein Expression Amount 2 ug 400 ng 80 ng 16 ng 3.2 ng 640pg 128 pg 26 Transfected pg Protein 16.7 21.8 16.4 4.7 0 0 0 0 (mU/I)

Example 169 In Vitro Expression of Insulin Glulisine Modified mRNA

The cells were transfected with Insulin Glulisine modified mRNA (mRNAsequence shown in SEQ ID NO: 21469; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap) fully modified with5-methylcytosine and pseudouridine (5mC/pU), fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/lmpU), modified where25% of uridine modified with 2-thiouridine and 25% of cytosine modifiedwith 5-methylcytosine (s2U and 5mC), fully modified with pseudouridine(pU), or fully modified with 1-methylpseudouridine (1 mpU) which hadbeen complexed with Lipofectamine-2000 from Invitrogen (Carlsbad,Calif.) at the concentration shown in Table 211. The total volume oftransfection mix per 96-well was 50 μl, split in two aliquots 25 μleach. One 25 μl aliquot contained 1 μl of respective mmRNA and 24 μlOpti-MEM, the other 25 μl aliquot contained 0.4 μl Lipofectamine-2000and 24.6 μl Opti-MEM. Where needed, the aliquot containing modified mRNAand Opti-MEM was serially diluted in Opti-MEM. Both aliquots wereincubated separately for 15 min at room temperature. Subsequently, bothaliquots were mixed together and incubated for another 20 min at roomtemperature before finally transferring the total 50 μl transfection mixto a 96-well containing 100 μl cell culture medium plus 35000 HEK293cells. Cells were then incubated for 24 h in a humidified 37° C./5% CO₂cell culture incubator before harvesting the cell culture supernatant orpreparing cell lysate. The protein expression was detected by ELISA andthe protein (pg/ml) is shown in FIG. 52 and in Table 211. In Table 211,“>” means greater than.

TABLE 211 Protein Expression Amount 2 ug 400 ng 80 ng 16 ng 3.2 ng 640pg 128 pg 26 Transfected pg Protein >100 >100 >100 95.9 13.1 0 0 0(mU/I)

Example 170 Dose Response and Injection Site Selection and Timing

To determine the dose response trends, impact of injection site andimpact of injection timing, studies are performed following the protocoloutlined in Example 35. In these studies, varied doses of 1 ug, 5 ug, 10ug, 25 ug, 50 ug, and values in between are used to determine doseresponse outcomes. Split dosing for a 100 ug total dose includes threeor six doses of 1.6 ug, 4.2 ug, 8.3 ug, 16.6 ug, or values and totaldoses equal to administration of the total dose selected.

Injection sites are chosen from the limbs or any body surface presentingenough area suitable for injection. This may also include a selection ofinjection depth to target the dermis (Intradermal), epidermis(Epidermal), subcutaneous tissue (SC) or muscle (IM). Injection anglewill vary based on targeted delivery site with injections targeting theintradermal site to be 10-15 degree angles from the plane of the surfaceof the skin, between 20-45 degrees from the plane of the surface of theskin for subcutaneous injections and angles of between 60-90 degreeesfor injections substantially into the muscle.

Example 171 Protein Production of Transforming Growth Factor Beta 1 inHeLa Cell Supernatant

The day before transfection, 100,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 250 ng oftransforming growth factor beta 1 (TGFB1) modified RNA (mRNA sequenceshown in SEQ ID NO: 1668; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (5mC/pU), fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/lmpU), modified where25% of uridine modified with 2-thiouridine and 25% of cytosine modifiedwith 5-methylcytosine (s2U and 5mC), fully modified with pseudouridine(pU), or fully modified with 1-methylpseudouridine (1 mpU), was dilutedin 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island,N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) wasused as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM. After 5 minutes of incubationat room temperature, both solutions were combined and incubated anadditional 15 minute at room temperature. Then the 20ul combinedsolution was added to the 100 ul cell culture medium containing the HeLacells and incubated at room temperature. A control of lipofecatmine 2000treated cells (L2000) and untreated cells was also analyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing TGFB1 was collected and centrifuged at 10.000 rcf for 2minutes. The cleared supernatants were then analyzed with an TGFB1 ELISAkit (R & D Systems, Minneapolis, Minn.) according to the manufacturerinstructions. All samples were diluted until the determined values werewithin the linear range of the ELISA standard curve. The amount ofprotein produced is shown in Table 212.

TABLE 212 Protein Production TGFB1 Sample (pg/ml) 5-methylcytosine andpseudouridine (5mC and pU) 704 5-methylcytosine and 1-methylpsudouridine(5mC and 1mpU) 1800 25% of uridine modified with 2-thiouridine and 25%of 57 cytosine modified with 5-methylcytosine (s2U and 5mC)Pseudouridine (pU) 743 1-methylpseudouridine (1mpU) 2279 L2000 4Untreated 0

Example 172 Protein Production of Transforming Growth Factor Beta 1 inHeLa Cell Supernatant

The day before transfection, 100,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 250 ng oftransforming growth factor beta 1 (TGFB1) modified RNA (mRNA sequenceshown in SEQ ID NO: 1668; polyA tail of approximately 140 nucleotidesnot shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine (5mC/pU), fully modified with5-methylcytosine and 1-methylpseudouridine (5mC/1mpU), modified where25% of uridine modified with 2-thiouridine and 25% of cytosine modifiedwith 5-methylcytosine (s2U and 5mC), fully modified with pseudouridine(pU), or fully modified with 1-methylpseudouridine (1 mpU), was dilutedin 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island,N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) wasused as transfection reagent and 0.2 ul of lipofectamine 2000 wasdiluted to 10 ul final volume of OPTI-MEM. After 5 minutes of incubationat room temperature, both solutions were combined and incubated anadditional 15 minute at room temperature. Then the 20 ul combinedsolution was added to the 100 ul cell culture medium containing the HeLacells and incubated at room temperature. A control of lipofecatmine 2000treated cells (L2000) and untreated cells was also analyzed.

After an 18 to 22 hour incubation cell culture lysates of cellsexpressing TGFB1 was collected and centrifuged at 10.000 rcf for 2minutes. The cell lysates (lug per ELISA) were then analyzed with anTGFB1 ELISA kit (R & D Systems, Minneapolis, Minn.) according to themanufacturer instructions. All samples were diluted until the determinedvalues were within the linear range of the ELISA standard curve. Theamount of protein produced is shown in Table 213.

TABLE 213 Protein Production TGFB1 Sample (pg/ml) 5-methylcytosine andpseudouridine (5mC and pU) 6214 5-methylcytosine and1-methylpsudouridine (5mC and 1mpU) 9257 25% of uridine modified with2-thiouridine and 25% of 5929 cytosine modified with 5-methylcytosine(s2U and 5mC) Pseudouridine (pU) 5157 1-methylpseudouridine (1mpU) 10857L2000 0 Untreated 1043

Example 173 Confirmation of Peptide Identity from Modified mRNA

Cell lysates containing protein produced from angiotensin-covertingenzyme 2 (ACE2) modified mRNA (mRNA sequence shown in SEQ ID NO: 21678;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1), activity-dependent neuroprotector (ADNP) modified mRNA (mRNAsequence shown in SEQ ID NO: 21679; polyA tail of approximately 140nucleotides not shown in sequence; 5′ cap, Cap1), Septin-4 (ARTS)modified mRNA (mRNA sequence shown in SEQ ID NO: 21680; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1), bonemorphogenetic protein 2 (BMP2) modified mRNA (mRNA sequence shown in SEQID NO: 21681; polyA tail of approximately 140 nucleotides not shown insequence; 5′ cap, Cap1), ceruloplasmin (CP) modified mRNA (mRNA sequenceshown in SEQ ID NO: 1620; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1), COMM domain-containing protein 1(COMMD1) modified mRNA (mRNA sequence shown in SEQ ID NO: 21682; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1), diablo, IAP-binding mitochondrial protein (DIABLO or SMAC)modified mRNA (mRNA sequence shown in SEQ ID NO: 21683; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),coagulation factor XIII alpha (F13A1) modified mRNA (mRNA sequence shownin SEQ ID NO: 21684; polyA tail of approximately 160 nucleotides notshown in sequence; 5′ cap, Cap1), Hepcidin (HEPC) modified mRNA (mRNAsequence shown in SEQ ID NO: 21471; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1), IgM Heavy Chainmodified mRNA (mRNA sequence shown in SEQ ID NO: 21685; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1),Interleukin-21 (IL21) modified mRNA (mRNA sequence shown in SEQ ID NO:21686; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1), N-acetylglutamate synthase (NAGS) modified mRNA(mRNA sequence shown in SEQ ID NO: 21687; polyA tail of approximately160 nucleotides not shown in sequence; 5′ cap, Cap1) orE3ubiquitin-protein ligase (XIAP) modified mRNA (mRNA sequence shown inSEQ ID NO: 21688; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1),fully modified with 5-methylcytosine andpseudouridine (5mC and pU), fully modified with 5-methylcytosine and1-methylpsudouridine (5mC and lmpU), modified where 25% of uridinemodified with 2-thiouridine and 25% of cytosine modified with5-methylcytosine (s2U and 5mC), fully modified with pseudouridine (pU),or fully modified with 1-methylpseudouridine (1 mpU) were evaluatedusing the LC-MS/MS with quantitative LC-MRM as described in Example 157.Peptide fragments identified for the evaluated proteins are shown inTable 214

TABLE 214 Protein and Peptide Fragment Sequences Peptide Fragment 5mC5mC s2U SEQ ID and and and NO pU 1mpU 5mC pU 1mpU ACE2 ISFNFFVTAPK 21689— Yes — Yes Yes LWAWESWR 21690 — Yes Yes Yes Yes SEPWTLALENVVG 21691 —Yes Yes Yes Yes AK ADNP NVHSEDFENR 21692 — — — — Yes ARTSESGTDFPIPAVPPGT 21693 Yes Yes Yes Yes Yes DPETEK FLEDTTDDGELSK 21694 YesYes Yes Yes Yes HAVDIEEK 21695 Yes Yes Yes Yes Yes BMP2 NYQDMVVEGCGCR21696 — Yes Yes Yes Yes SFHHEESLEELPETS 21697 — Yes Yes Yes Yes GKWESFDVTPAVMR 21698 Yes Yes Yes Yes Yes CP ALYLQYTDETFR 21699DIASGLIGPLIICK 21700 GAYPLSIEPIGVR 21701 COMMD1 ESLMNQSR 21702 Yes YesYes Yes Yes HSAQIHTPVAIIELE 21703 — — Yes Yes Yes LGK WNSGLR 21704 — YesYes Yes Yes DIABLO AVYTLTSLYR 21705 Yes Yes Yes Yes Yes LAEAQIEELR 21706Yes Yes Yes Yes Yes NHIQLVK 21707 Yes Yes Yes Yes Yes F13A1MYVAVWTPYGVLR 21708 Yes Yes Yes Yes Yes STVLTIPEIIIK 21709 — Yes Yes YesYes VEYVIGR 21710 — — Yes Yes Yes HEPC DTHFPIBIFBBGBBHR 21711 Yes YesYes Yes Yes IgM Heavy Chain TSTSPIVK 21712 — Yes — Yes Yes WIYDTSK 21713— Yes Yes Yes Yes WKIDGSER 21714 Yes Yes Yes Yes Yes IL21 IINVSIK 21715Yes Yes — Yes Yes LTBPSBDSYEK 21716 Yes Yes Yes Yes Yes QLIDIVDQLK 21717Yes Yes Yes Yes Yes NAGS DLQTLFWR 21718 — — — — Yes DSYELVNHAK 21719 YesYes Yes Yes Yes HNAAAAVPFFGGG 21720 Yes Yes Yes Yes Yes SVLR XIAPAGFLYTGEGDTVR 21721 Yes Yes Yes Yes Yes DHFALDRPSETHAD 21722 Yes — — YesYes YLLR IFTFGTWIYSVNK 21723 Yes Yes Yes Yes Yes

Example 174 Protein Expression Confirmation

Modified mRNA fully modified with 5-methylcytosine and pseudouridine(5mC/pU), fully modified with 5-methylcytosine and 1-methylpseudouridine(5mC/lmpU), modified where 25% of the cytosines replaced with5-methylcytosine and 25% of the uridines replaced with 2-thiouridine(s2U/5mC), fully modified with 1-methylpseudouridine (1 mpU) or fullymodified with pseudouridine (pU) encoding the proteins described inTable 215 were analyzed by fluorescence and/or western blot in order toconfirm the expression of protein and the length of the proteinexpressed. Table 215 describes the sequence of the modified mRNA (polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1), the chemical modification evaluated and the length (in basepairs), if known.

TABLE 215 Protein and Expression Modified mRNA SEQ ID 5mC/ 5mC/ s2U/Protein NO pU 1mpU 5mC pU 1mpU Glucagon 21724 — Yes Yes Yes — (GLP1) 451 bp  451 bp  451 bp Glycoprotein 21725 — Yes Yes Yes — hormones, 634 bp  634 bp  634 bp alpha polypeptide (FSH-alpha) Surfactant 21726Yes — — — — protein B 1432 bp (SFTPB) Anti-tumor 21727 — Yes Yes Yes —necrosis factor 1684 bp 1684 bp 1684 bp alpha Sortilin 1 21728 Yes — — —— (SORT1) 2782 bp ImG Light 21729 Yes — — — — Chain  982 bp Sirtuin 121730 Yes — — — — (SIRT1) 2530 bp galactosamine 21731 — Yes Yes Yes Yes(N-acetyl)-6- 1855 bp 1855 bp 1855 bp 1855 sulfate bp sulfatase (GALNS)Complement 21732 Yes — — — — factor H (CFH) 3982 bp colony 21733 Yes — —— — stimulating  854 bp factor 2 (granulocyte- macrophage) (GM-CSF)Klotho (KL) 21734 Yes — — — — 1933 bp coagulation 21684 Yes — — — —factor XIII 2485 bp alpha (F13A1) Phosphorylase 21735 Yes — — — —kinase, alpha 2 3994 bp (PHKA2) Phosphorylase 21736 Yes — — — — kinase,beta 3568 bp Tyrosinase 21737 — Yes — Yes — (TYR) 1876 bp 1876 bpangiotensin- 21678 Yes — — — — coverting 2704 bp enzyme 2 (ACE2)neuregulin 1 21738 — Yes Yes Yes — 2209 bp 2209 bp 2209 bp activity-21679 Yes — — — — dependent 3595 bp neuroprotector (ADNP) Interleukin-2121686 Yes — — — — (IL21)  754 bp COMM 21682 Yes — — — — domain-  859 bpcontaining protein 1 (COMMD1)

Example 175 Intranasal Lung Delivery of 1-methylpseudouridine or5-methylcytosine and 1-methylpseudouridine Modified Luciferase mRNAFormulated in Cationic Lipid Nanoparticle

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) fully modified with 1-methylpseudouridine (Luc-G5-LNP-KC2) orfully modified with 1-methylpseudouridine and 5-methylcytosine(Luc-G2-LNP-KC2) were formulated in the cationic Lipid Nanoparticle(LNP-KC2) as described in Table 216.

TABLE 216 Formulation Formulation NPA-127-1 NPA-135-1 Lipid DLin-KC2-DMADLin-KC2-DMA Lipid/mRNA 20:1 20:1 ratio (wt/wt) Mean Size 114 nm 95 nmPDI: 0.10 PDI: 0.11 Zeta at pH 7.4 −0.5 mV −1.0 mV Encaps. (RiboGr) 77%100%

The formulations were administered intranasally (I.N.) to Balb-C mice ata dose of 0.3 mg/kg. Twenty minutes prior to imaging, mice were injectedintraperitoneally with a D-luciferin solution at 150 mg/kg. Animals werethen anesthetized and images were acquired with an IVIS Lumina IIimaging system (Perkin Elmer). Bioluminescence was measured as totalflux (photons/second) of the entire mouse. The mice were imaged at 2hours, 8 hours, 48, and 72 hours after dosing and the average total flux(photons/second) was measured and shown in Table 217. The backgroundflux was about 6.3+05 p/s. The results of the imaging of Luc-G5-LNP-KC2or Luc-G5-LNP-KC2 vs. Vehicle are shown in the Table 217, “NT” means nottested.

TABLE 217 Luciferase expression after intranasal dosing Luc-G5- Luc-G2-Vehicle LNP-KC2 LNP-KC2 (PBS) Route Time Point Flux (p/s) Flux (p/s)Flux (p/s) I.N. 2 hrs 1.53E+06 5.81E+05 3.87E+05 I.N. 8 hrs 1.09E+079.35E+05 5.57E+05 I.N. 24 hrs 5.93E+06 5.33E+05 4.89E+05 I.N. 28 hrs1.22E+06 7.63E+05 8.81E+05 I.N. 72 hrs 8.43E+05 NT 8.17E+05

Example 176 Intranasal Lung Delivery of 1-methylpseudouridine or5-methylcytosine and 1-methylpseudouridine Modified Luciferase mRNAlipoplexed in Lipofectamine 2000

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) fully modified with 1-methylpseudouridine (Luc-G5-Lipoplex) orfully modified with 1-methylpseudouridine and 5-methylcytosine(Luc-G2-Lipoplex) were lipoplexed in two steps, first step was thedilution of 16.6 ul from 3 mg/ml of G5 or G2 modified luciferase mRNA in108.5 ul DMEM and the second step was the dilution of 100 ul LF2000 in25 ul DMEM then both mixed together gently to make 250 ul total mixerwhich incubated 5-10 min at RT to form a Lipid-mRNA complex beforeinjection. The lipoplex from both Luciferase mRNA fully modified with1-methylpseudouridine or 1-methylpseudouridine and 5-methylcytosine wereadministered intranasally (I.N.) to Balb-C mice at a dose of 0.5 mg/kg.

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredand is shown in Table 218. The background signal was about 4.8+05 p/s.The background flux was about 6.3+05 p/s. The results of the imaging ofLuc-G5-Lipoplex or Luc-G2-Lipoplex vs. Vehicle are shown in the Table218.

TABLE 218 Luciferase expression after intranasal dosing Luc-G5- Luc-G2-Vehicle Lipoplex Lipoplex (PBS) Route Time Point Flux (p/s) Flux (p/s)Flux (p/s) I.N. 2 hrs 8.37E+05 9.58E+05 3.87E+05 I.N. 8 hrs 8.42E+057.11E+05 5.57E+05 I.N. 24 hrs  5.74E+05 5.53E+05 4.89E+05

Example 177 Intranasal Lung Delivery of 1-methylpseudouridine or5-methylcytosine and 1-methylpseudouridine Modified Luciferase mRNAFormulated in PBS

Luciferase modified mRNA (mRNA sequence shown in SEQ ID NO: 21446; polyAtail of approximately 140 nucleotides not shown in sequence; 5′ cap,Cap1) fully modified with 1-methylpseudouridine (Luc-G5-Buffer(PBS)) orfully modified with 1-methylpseudouridine and 5-methylcytosine(Luc-G2-Buffer(PBS)) formulated in PBS (pH 7.4) were administeredintranasally (I.N.) to Balb-C mice at a dose of 7.5 mg/kg.

Twenty minutes prior to imaging, mice were injected intraperitoneallywith a D-luciferin solution at 150 mg/kg. Animals were then anesthetizedand images were acquired with an IVIS Lumina II imaging system (PerkinElmer). Bioluminescence was measured as total flux (photons/second) ofthe entire mouse. The mice were imaged at 2 hours, 8 hours and 24 hoursafter dosing and the average total flux (photons/second) was measuredand is shown in Table 219. The background flux was about 4.8+05 p/s. Thebackground flux was about 6.3+05 p/s. The results of the imaging ofLuc-G5-in Buffer vs. Vehicle are shown in the Table 219.

TABLE 219 Luciferase expression after intranasal dosing Luc-G5- Luc-G2-Vehicle in Buffer (PBS) in Buffer (PBS) (PBS) Route Time Point Flux[p/s] Flux [p/s] Flux (p/s) I.N. 2 hrs 4.50E+05 9.58E+05 3.87E+05 I.N. 8hrs 7.12E+05 7.11E+05 5.57E+05 I.N. 24 hrs  4.47E+05 5.53E+05 4.89E+05

Example 178 In Vitro Transfection of Interleukin 7

Interleukin 7 (IL7) modified mRNA (mRNA sequence shown in SEQ ID NO:1656; polyA tail of approximately 160 nucleotides not shown in sequence;5′ cap, Cap1) was transfected via reverse transfection in HumanKeratinocyte cells in 24 multi-well plates. Human Keratinocytes cellswere grown in EPILIFE® medium with Supplement S7 from Invitrogen(Carlsbad, Calif.) until they reached a confluence of 50-70%. The cellswere transfected with 0, 46.875, 93.75, 187.5, 375 and 750, ng ofmodified mRNA (mmRNA) encoding IL-7 which had been complexed withRNAIMAX™ from Invitrogen (Carlsbad, Calif.). The RNA:RNAIMAX™ complexwas formed by first incubating the RNA with Supplement-free EPILIFE®media in a 5× volumetric dilution for 10 minutes at room temperature. Ina second vial, RNAIMAX™ reagent was incubated with Supplement-freeEPILIFE® Media in a 10× volumetric dilution for 10 minutes at roomtemperature. The RNA vial was then mixed with the RNAIMAX™ vial andincubated for 20-30 minutes at room temperature before being added tothe cells in a drop-wise fashion.

The fully optimized mRNA encoding IL-7 (mRNA sequence shown in SEQ IDNO: 1656; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) was fully modified with 5-methylcytosine andpseudouridine. Cells were transfected with the mmRNA encoding IL-7 andIL-7 concentration (ρg/ml) in the culture medium was measured at 6 and12 hours post-transfection for each of the concentrations using an ELISAkit from Invitrogen (Carlsbad, Calif.) following the manufacturersrecommended instructions. These data, shown in FIG. 53, show thatmodified mRNA encoding IL-7 is capable of being translated in HumanKeratinocyte cells at the 375 and 750 ng doses.

Example 179 In Vitro Transfection of Erythropoietin

Erythropoietin (EPO) modified mRNA (mRNA sequence shown in SEQ ID NO:1638; polyA tail of approximately 160 nucleotides not shown in sequence;5′ cap, Cap1) was transfected via reverse transfection in HumanKeratinocyte cells in 24 multi-well plates. Human Keratinocytes cellswere grown in EPILIFE® medium with Supplement S7 from Invitrogen(Carlsbad, Calif.) until they reached a confluence of 50-70%. The cellswere transfected with 46.875, 93.75, 187.5, 375 and 750, ng of modifiedmRNA (mmRNA) encoding EPO which had been complexed with RNAIMAX™ fromInvitrogen (Carlsbad, Calif.). The RNA:RNAIMAX™ complex was formed byfirst incubating the RNA with Supplement-free EPILIFE® media in a 5×volumetric dilution for 10 minutes at room temperature. In a secondvial, RNAIMAX™ reagent was incubated with Supplement-free EPILIFE® Mediain a 10× volumetric dilution for 10 minutes at room temperature. The RNAvial was then mixed with the RNAIMAX™ vial and incubated for 20-30minutes at room temperature before being added to the cells in adrop-wise fashion.

The fully optimized mRNA encoding EPO (mRNA sequence shown in SEQ ID NO:1638; polyA tail of approximately 160 nucleotides not shown in sequence;5′ cap, Cap1) was fully modified with 5-methylcytosine andpseudouridine. Cells were transfected with the mmRNA encoding EPO andEPO concentration (ρg/ml) in the culture medium was measured at 6 and 12hours post-transfection for each of the concentrations using an ELISAkit from Invitrogen (Carlsbad, Calif.) following the manufacturersrecommended instructions. These data, shown in FIG. 54, show thatmodified mRNA encoding EPO is capable of being translated in HumanKeratinocyte cells.

Example 180 Detection of Lysosomal Acid Lipase Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 750 ng of lysosomal acid lipase (LAL)mRNA (mRNA sequence shown in SEQ ID NO: 1657; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine) was diluted in 250 ulfinal volume of OPTI-MEM® (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 5.5 ul were diluted in 250 ul final volume ofOPTI-MEM®. After 5 minutes of incubation at room temperature, bothsolutions were combined and incubated an additional 15 minutes at roomtemperature. Then 500 ul of the combined solution was added to the 3 mlcell culture medium containing the HeLa cells. The plates were thenincubated as described before.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4×SDS loading buffer (both from Life Technologies, GrandIsland, N.Y.). Samples were heated at 95° C. for 5 minutes and loaded onthe gel. Standard settings were chosen by the manufacturer, 200V, 120 mAand maximum 25 W. Run time was 60 minutes, but no longer than runningdye reaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. Primary antibodyanti-lysosomal acid lipase antibody; Abcam Cat No. ab89771) against thetarget protein was applied in 3 ml of 5% BSA in 1× TBS solution at a1:500 to 1:2000 dilution for 3 hours at room temperature and gentleagitation on an orbital shaker. Membranes are washed 3 times with lxTBS/0.1% Tween, 5 minutes each time with gentle agitation. The secondaryantibody ((Western Breeze, Invitrogen) was conjugated to horse radishperoxidase and binds to the primary antibody. The secondary antibody wasdiluted of 1:1000 to 1:5000 in 5% BSA in lx TBS and incubated for 3hours at room temperature. As shown in FIG. 55 the western blotdetection of LAL was close to 45.4 kDa. In FIG. 55, lanes 1 and 2 showthe modified mRNA and lanes 3 and 4 show the dosing vehicle only.

Example 181 Detection of Glucocerebrosidase Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 750 ng of glucocerebrosidase mRNA (mRNAsequence shown in SEQ ID NO: 1645; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine was diluted in 250 ul final volume ofOPTI-MEM® (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 5.5 ul were diluted in 250 ul final volume of OPTI-MEM®. After 5minutes of incubation at room temperature, both solutions were combinedand incubated an additional 15 minutes at room temperature. Then 500 ulof the combined solution was added to the 3 ml cell culture mediumcontaining the HeLa cells. The plates were then incubated as describedbefore.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4×SDS loading buffer (both from Life Technologies, GrandIsland, N.Y.). Samples were heated at 95° C. for 5 minutes and loaded onthe gel. Standard settings were chosen by the manufacturer, 200V, 120 mAand maximum 25W. Run time was 60 minutes, but no longer than running dyereaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. Primary antibody(Anti-GBA antibody; Abcam Cat No. ab55080 and Sigma Cat No. G4046)against the target protein were applied in 3 ml of 5% BSA in 1× TBSsolution at a 1:500 to 1:2000 dilution for 3 hours at room temperatureand gentle agitation on an orbital shaker. Membranes are washed 3 timeswith 1×TBS/0.1% Tween, 5 minutes each time with gentle agitation. Thesecondary antibody (Western Breeze, Invitrogen) was conjugated to horseradish peroxidase and binds to the primary antibody. The secondaryantibody was diluted of 1:1000 to 1:5000 in 5% BSA in 1× TBS andincubated for 3 hours at room temperature. As shown in FIG. 56 thewestern blot detection of glucocerebrosidase was close to 59.7 kDa. InFIG. 56, lanes 1 and 2 show the modified mRNA and lanes 3 and 4 show thedosing vehicle only.

Example 182 Detection of Iduronate 2-Sulfatase Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 750 ng of iduronate 2-sulfatase mRNA(mRNA sequence shown in SEQ ID NO: 1652; polyA tail of approximately 160nucleotides not shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine) was diluted in 250 ul final volumeof OPTI-MEM® (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 5.5 ul were diluted in 250 ul final volume of OPTI-MEM®. After 5minutes of incubation at room temperature, both solutions were combinedand incubated an additional 15 minutes at room temperature. Then 500 ulof the combined solution was added to the 3 ml cell culture mediumcontaining the HeLa cells. The plates were then incubated as describedbefore.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4× SDS loading buffer (both from Life Technologies,Grand Island, N.Y.). Samples were heated at 95° C. for 5 minutes andloaded on the gel. Standard settings were chosen by the manufacturer,200V, 120 mA and maximum 25W. Run time was 60 minutes, but no longerthan running dye reaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. Primary antibodies(anti-iduronate 2 sulfatase antibody; Abcam Cat No. ab 70025 and R&DSystems Cat No. AF2449) against the target protein were applied in 3 mlof 5% BSA in 1× TBS solution at a 1:500 to 1:2000 dilution for 3 hoursat room temperature and gentle agitation on an orbital shaker. Membranesare washed 3 times with 1× TBS/0.1% Tween, 5 minutes each time withgentle agitation. The secondary antibody (Western Breeze, Invitrogen)was conjugated to horse radish peroxidase and binds to the primaryantibody. The secondary antibody was diluted of 1:1000 to 1:5000 in 5%BSA in 1× TBS and incubated for 3 hours at room temperature. As shown inFIG. 57 the western blot detection of iduronate 2-sulfatase was close to76 kDa. In FIG. 57, lanes 1 and 2 show the modified mRNA and lanes 3 and4 show the dosing vehicle only.

Example 183 Detection of Luciferase Protein: Western Blot

The day before transfection, 750,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment withTrypsin-Ethylenediaminetetraacetic acid (EDTA) solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of 3ml Eagle's minimal essential medium (EMEM) (supplemented with 10% fetalcalf serum (FCS) and 1× Glutamax) per well in a 6-well cell cultureplate (Corning, Manassas, Va.). The cells were grown at 37° C. in 5% CO₂atmosphere overnight. Next day, 750 ng of luciferase mRNA (mRNA sequenceshown in SEQ ID NO: 21446; polyA tail of approximately 160 nucleotidesnot shown in sequence; 5′ cap, Cap1) fully modified with5-methylcytosine and pseudouridine) was diluted in 250 ul final volumeof OPTI-MEM® (LifeTechnologies, Grand Island, N.Y.). Lipofectamine 2000(LifeTechnologies, Grand Island, N.Y.) was used as transfection reagentand 5.5 ul were diluted in 250 ul final volume of OPTI-MEM®. After 5minutes of incubation at room temperature, both solutions were combinedand incubated an additional 15 minutes at room temperature. Then 500 ulof the combined solution was added to the 3 ml cell culture mediumcontaining the HeLa cells. The plates were then incubated as describedbefore.

After an 16-18 hour incubation, the medium was removed and the cellswere washed with 1 ml PBS. After complete removal of the PBS, 500 ul offresh PBS was added. Cells were harvested by scraping with a cellscraper. Harvested cells receiving the same mRNA were then combined inone 1.5 ml Eppendorf tube.

Cells were pelleted by centrifugation at 3,000 rpm for 2 minutes. ThePBS was removed and cells lysed in 250 ul radio immunoprecipitatoinassay (RIPA) buffer (containing PMSF and eukaryotic protease inhibitormix) (all from BostonBioProducts, Ashland, Mass.) by gentle pipetting.Lysates were cleared by centrifugation at 10,000 rpm at 4° G for 10minutes. Cleared lysates were transferred to Amicon filters with 10,000kd molecular cutoff (Waters, Milford, Mass.) and spun at 12,000 rpm and4° G for 20 minutes. The concentrated protein lysate was recovered byplacing the inverted filter in a fresh 1.5 ml Eppendorf tube andspinning at 3,000 rpm for 1 minute. The final volume of the lysate isbetween 25-40 ul.

Protein concentration was determined using the BCA kit formicrotiter-plates from Pierce (Thermo Fisher, Rockford, Ill.). Thestandard proteins of the titration curve were dissolved in RIPA buffer(as described for cell lysate generation) and not in dilution buffer.

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 ul, 4 ul of 10× reducingagent and 10 ul 4× SDS loading buffer (both from Life Technologies,Grand Island, N.Y.). Samples were heated at 95° C. for 5 minutes andloaded on the gel. Standard settings were chosen by the manufacturer,200V, 120 mA and maximum 25W. Run time was 60 minutes, but no longerthan running dye reaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1x tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. Primary antibody(anti-luciferase, AbCam Cat No. ab21176) against the target protein wasapplied in 3 ml of 5% BSA in 1× TBS solution at a 1:500 to 1:2000dilution for 3 hours at room temperature and gentle agitation on anorbital shaker. Membranes are washed 3 times with 1× TBS/0.1% Tween, 5minutes each time with gentle agitation. The secondary antibody (WesternBreeze, Invitrogen) was conjugated to horse radish peroxidase and bindsto the primary antibody. The secondary antibody was diluted of 1:1000 to1:5000 in 5% BSA in 1× TBS and incubated for 3 hours at roomtemperature. As shown in FIG. 58 the western blot detection ofluciferase was close to 60.7 kDa. In FIG. 58, lanes 1 and 2 show themodified mRNA and lanes 3 and 4 show the dosing vehicle only.

Example 184 Herceptin In Vivo Studies

Herceptin heavy chain (HC) modified mRNA (SEQ ID NO: 21451; polyA tailof approximately 140 nucleotides not shown in sequence; 5′ cap, Cap1;fully modified with 5-methylcytosine and 1-methylpseudouridine) andHerceptin light chain (LC) modified mRNA (SEQ ID NO: 21452; polyA tailof approximately 140 nucleotides not shown in sequence; 5′ cap, Cap1;fully modified with 5-methylcytosine and 1-methylpseudouridine) wereformulated in DLin-MC3-DMA and DLin-KC2-DMA as described in Table 220.

TABLE 220 DLin-MC3-DMA and DLin-KC2-DMA Formulations FormulationNPA-159-1 Herceptin HC:LC  2:1 Ratio (wt/wt) Lipid DLin-KC2-DMALipid/Total mRNA 20:1 ratio (wt/wt) Mean Size 100 nm PDI: 0.10 Zeta atpH 7.4 1.9 mV Encaps. (RiboGr) 100%

The Herceptin formulations described in Table X1 were administeredintravenously to mice (CD1) at a dose of 0.5 mgs/kg. A control ofLuciferase mRNA was also evaluated (Luc in FIG. 59). The mice were bledat 8 hours, 24 hours, 48 hours, 96 hours, 168 hours (7 days), 336 hours(14 days), 504 hours (21 days) after the administration of Herceptin todetermine the serum IgG concentration by ELISA (total human IgGdetection kit by ABCAM®, Cambridge Mass.). The control Luciferaseinjected animals were bled at only 8 hr time point. The results areshown in Table 222 and FIG. 59.

TABLE 222 Herceptin Formulations Injection Amount of Post dose HumanVolume modified Dose collection IgG mRNA Route (ul) RNA (ug) (mg/kg)(Hrs) (ng/ml) Herceptin IV 100 10 0.5 8 39.4 (LC + HC) Herceptin IV 10010 0.5 24 56.1 (LC + HC) Herceptin IV 100 10 0.5 48 43.8 (LC + HC)Herceptin IV 100 10 0.5 96 56.7 (LC + HC) Herceptin IV 100 10 0.5 16831.7 (LC + HC) Herceptin IV 100 10 0.5 336 17.6 (LC + HC) Herceptin IV100 10 0.5 504 3 (LC + HC) Luciferase IV 100 10 0.5 8 0.57

Example 185 Herceptin In Vitro Studies

The day before transfection, 15,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. The next day, 150 ng ofthe herceptin heavy chain (HC) mRNA (SEQ ID NO: 21451; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and 1-methylpsuedouridine) or 75 ngHerceptin light chain (LC) (SEQ ID NO: 21452; polyA tail ofapproximately 140 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and 1-methylpsuedouridine) or 500 ng ofthe Herceptin HC and 250 ng of Herceptin LC together was diluted in 10ul final volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 0.2 ul of lipofectamine 2000 was diluted to 10ul final volume of OPTI-MEM. After 5 minutes of incubation at roomtemperature, both solutions were combined and incubated an additional 15minute at room temperature. Then the 20 ul combined solution was addedto the 100 ul cell culture medium containing the HeLa cells andincubated at room temperature. As controls, Recombinant human IgG (HuIgG ctrl), assay buffer and culture medium from untreated wells in thepresence absence of assay buffer (Assay buffer+Control Media) were alsoanalyzed.

After an 18 to 22 hour incubation cell culture supernatants of cellsexpressing Herceptin heavy or light chains were collected and analyzedwith the total human IgG ELISA kit (ABCAM®, Cambridge Mass.) accordingto the manufacturer instructions. The amount of IgG produced is shown inTable 223 and FIG. 60. In Table X3, “OTC” means off the chart.

TABLE 223 Herceptin Study Sample IgG (ng/ml) Recombinant Human IgG OTCHerceptin Heavy Chain 12 Herceptin Light Chain 0 Herceptin Heavy + LightChain 348 Assay buffer 9 Assay buffer + Control Media 12

Example 186 Herceptin In Vitro Studies: Confirmation of AntibodyProduction in Vitro by Immunoblotting

HeLa cells were transfected in vitro in absence of serum using theHerceptin heavy and light chain mRNAs (herceptin heavy chain (HC) mRNA(SEQ ID NO: 21451; polyA tail of approximately 140 nucleotides not shownin sequence; 5′ cap, Cap1; fully modified with 5-methylcytosine and1-methylpsuedouridine), Herceptin light chain (LC) (SEQ ID NO: 21452;polyA tail of approximately 140 nucleotides not shown in sequence; 5′cap, Cap1; fully modified with 5-methylcytosine and1-methylpsuedouridine) or Herceptin HC and LC together (H&L)) describedin Example 185. 24 hours after transfection, supernatants werecollected, concentrated and 4 ug of protein was loaded, in presence(Lanes 1-5 in FIG. 61) and absence (Lanes 6-10 in FIG. 61) of reducingagents, into each lane of a 4-12% MOPS NuPAGE gel for electrophoreticseparation. The proteins were then transferred onto a PVDF membrane andprobed using an antibody for simultaneous detection of heavy and lightIg chains.

As shown in FIG. 61, In the lanes where supernatants from the only lightchain transfected cells were run, light chain bands were detected underboth reducing and non-reducing conditions. In the non-reducing conditiona larger band was also detected at ˜50 kDa. This corroborates well withthe fact that light chain alone is secretory. The lanes containingsupernatants from only heavy chain mRNA transfected cells had no signalin presence or absence of reducing agents, confirming lack of secretionof the heavy chain alone. Supernatants from cells transfected with bothheavy and light chain mRNA had two bands in presence or absence ofreducing agents. In both conditions there was a light chain specificband. This represented light chain from denatured Ig molecule under thereducing condition and free light chain protein under the non-reducingcondition. Under the reducing condition, the heavy chain, which was onlysecreted in association with the light chain, was visible at 55 kDawhereas, under the non-reducing condition, no individual heavy chainband was apparent as expected. In that lane, a 160 kDa the total Ig bandwas clearly detectable. This confirms the molecular identity of thelight and heavy chain proteins produced when the chemically modifiedmRNAs encoding these proteins were transfected in HeLa cells.

In FIG. 61, “MWM” represents molecular weight marker, “LC” representsHerceptin light chain, “HC” represents Herceptin heavy chain, and“Media” represents culture media alone.

Example 187 Enzymatic Activity Assay

A. Glucocerebrosidase Enzyme Activity

HEK293 cells were transfected with 0, 625, 125, 250, 500, 1000 or 2000ng of glucocerebrosidase modified mRNA (mRNA sequence shown in SEQ IDNO: 1645; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andpseudouridine) complexed with RNAiMAX (Invitrogen). The cells werewashed with PBS at 6 hours, 12 hours, 24 hours and 48 hours aftertransfection. The cells were then lysed in 100 mM citrate-phosphatebuffer (pH 5.4) containing 0.54% sodium taurodeoxycholate with 1% TritonX-100. The cell lysate was then incubated in the presence of 0.5 mM of4-Methylumbelliferyl β-D-Galactopyranoside (4-MUG) substrate for 1 hourat 37° C. After an hour the reaction was stopped with an equivalentvolume of 200 mM sodium hydroxide. The conversion of 4-MU was measuredin the plate reader (Ex=370 nm; Em=460 nm). FIG. 62 shows the enzymaticactivity expressed as a percentage normalized against the mRNA controltransfection (iduronate 2-sulfatase (mRNA sequence shown in SEQ ID NO:1652; polyA tail of approximately 160 nucleotides not shown in sequence;5′ cap, Cap1) fully modified with 5-methylcytosine and pseudouridine)complexed with RNAiMAX (Invitrogen).

B. Lysosomal Acid Lipase Enzyme Activity

HEK293 cells were transfected with 0, 625, 125, 250, 500, 1000 or 2000ng of lysosomal acid lipase modified mRNA (mRNA sequence shown in SEQ IDNO: 1657; polyA tail of approximately 160 nucleotides not shown insequence; 5′ cap, Cap1) fully modified with 5-methylcytosine andpseudouridine) complexed with RNAiMAX (Invitrogen). The cells werewashed with PBS at 6 hours, 12 hours, 24 hours and 48 hours aftertransfection. The cells were then lysed in 100 mM citrate-phosphatebuffer (pH 5.4) containing 0.54% sodium taurodeoxycholate with 1% TritonX-100. The cell lysate was then incubated in the presence of 0.5 mM of4-Methylumbelliferyl f-3-D-Galactopyranoside (4-MUG) substrate for 1hour at 37° C. After an hour the reaction was stopped with an equivalentvolume of 200 mM sodium hydroxide. The conversion of 4-MU was measuredin the plate reader (Ex=370 nm; Em=460 nm). FIG. 63 shows the enzymaticactivity expressed as a percentage normalized against the mRNA controltransfection (iduronate 2-sulfatase (mRNA sequence shown in SEQ ID NO:1652; polyA tail of approximately 160 nucleotides not shown in sequence;5′ cap, Cap1) fully modified with 5-methylcytosine and pseudouridine)complexed with RNAiMAX (Invitrogen).

Example 188 In Vitro Transfection of Factor VIII: Protein Detection

Factor VIII modified mRNA (mRNA sequence shown in SEQ ID NO: 1623; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) was transfected via reverse transfection in HepG2 cells in 24multi-well plates. HepG2 cells were grown in EPILIFE® medium withSupplement S7 from Invitrogen (Carlsbad, Calif.) until they reached aconfluence of 50-70%. The cells were transfected with 0, 250 ng, 500 ng,750 ng, 1000 ng of modified mRNA (mmRNA) encoding Factor VIII which hadbeen complexed with RNAIMAX™ from Invitrogen (Carlsbad, Calif.). TheRNA:RNAIMAX™ complex was formed by first incubating the RNA withSupplement-free EPILIFE® media in a 5× volumetric dilution for 10minutes at room temperature. In a second vial, RNAIMAX™ reagent wasincubated with Supplement-free EPILIFE® Media in a 10× volumetricdilution for 10 minutes at room temperature. The RNA vial was then mixedwith the RNAIMAX™ vial and incubated for 20-30 minutes at roomtemperature before being added to the cells in a drop-wise fashion.

The fully optimized mRNA encoding Factor VIII (mRNA sequence shown inSEQ ID NO: 1623; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1) transfected with the HepG2 cells includedmodifications during translation such as natural nucleosidetriphosphates (NTP), pseudouridine at each uridine site and5-methylcytosine at each cytosine site (pseudo-U/5mC), andN1-methyl-pseudouridine at each uridine site and 5-methylcytosine ateach cytosine site (N1-methyl-Pseudo-U/5mC). Cells were transfected withthe mmRNA encoding Factor VIII and secreted Factor VIII concentration(ρg/ml) in the culture medium was measured at 18 hours post-transfectionfor each of the concentrations using an ELISA kit from Invitrogen(Carlsbad, Calif.) following the manufacturers recommended instructions.These data, shown in Table 224 and FIG. 64, show that modified mRNAencoding Factor VIII is capable of being translated in HepG2 cells.

TABLE 224 Factor VIII Dosing and Protein Secretion Dose (ng) 18 hours(IU/ml) Factor VIII Dose Containing Natural NTPs 0 0 250 0 500 0.4 7501.9 1000 2.1 Factor VIII Dose Containing Pseudo-U/5mC 0 0 250 7.8 5008.1 750 6.1 1000 7.8 Factor VIII Dose Containing N1-methy-Pseudo-U/5mC 00 250 3.5 500 4.6 750 6.2 1000 6.0

Example 189 In Vitro Transfection of Factor VIII: Chromogenic Activity

Factor VIII modified mRNA (mRNA sequence shown in SEQ ID NO: 1623; polyAtail of approximately 160 nucleotides not shown in sequence; 5′ cap,Cap1) was transfected via reverse transfection in HepG2 cells in 24multi-well plates. HepG2 cells were grown in EPILIFE® medium withSupplement S7 from Invitrogen (Carlsbad, Calif.) until they reached aconfluence of 50-70%. The cells were transfected with 0, 250 ng, 500 ng,750 ng, 1000 ng of modified mRNA (mmRNA) encoding Factor VIII which hadbeen complexed with RNAIMAX™ from Invitrogen (Carlsbad, Calif.). TheRNA:RNAIMAX™ complex was formed by first incubating the RNA withSupplement-free EPILIFE® media in a 5× volumetric dilution for 10minutes at room temperature. In a second vial, RNAIMAX™ reagent wasincubated with Supplement-free EPILIFE® Media in a 10× volumetricdilution for 10 minutes at room temperature. The RNA vial was then mixedwith the RNAIMAX™ vial and incubated for 20-30 minutes at roomtemperature before being added to the cells in a drop-wise fashion.

The fully optimized mRNA encoding Factor VIII (mRNA sequence shown inSEQ ID NO: 1623; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1) transfected with the HepG2 cells includedmodifications during translation such pseudouridine at each uridine siteand 5-methylcytosine at each cytosine site (pseudo-U/5mC), andN1-methyl-pseudouridine at each uridine site and 5-methylcytosine ateach cytosine site (N1-methyl-Pseudo-U/5mC). Cells were transfected withthe mmRNA encoding Factor VIII. When activated by thrombin, Factor VIIIforms an enzymatic complex with Factor IXa, phospholipids and Calcium,which activates Factor X to Factor Xa which activates Factor X, suppliedin the assay at a constant concentration and in excess, to Factor Xa.This activity is directly related to the amount of Factor VIII.

Generated Factor Xa was measured at 18 hours post-transfection by itsactivity on a specific Factor Xa chromogenic substrate (SXa-11),measured by the amount of pNA released, determined by color developmentat 405 nm in order to determine the activity of Factor VIII.

The Factor VIII activity was normalized to 25-30% Factor VIIIconcentrate control standard (% Activity). These data, shown in Table225 and FIG. 65, show that modified mRNA encoding Factor VIII is capableof being translated in HepG2 cells.

TABLE 225 Factor VIII Dosing and Protein Secretion Factor VIII DoseContaining Pseudo-U/5mC Dose (ng) 18 hours (% Activity) 0 0 250 31.7 50029.2 750 26.7 1000 20.4 Factor VIII Dose ContainingN1-methy-Pseudo-U/5mC Dose (ng) 18 hours (IU/ml) 0 0 250 25.8 500 30.4750 30.0 1000 26.7

Example 190 Low Density Lipoprotein Receptor (LDLR) Expression

A. In Vitro LDLR expression

Human embryonic kidney epithelial (HEK293) cells (LGC standards GmbH,Wesel, Germany) are seeded on 6-well plates (BD Biosciences, San Jose,USA). HEK293 are seeded at a density of about 500,000 cells per well in3 mL cell culture medium. Formulations containing LDLR mRNA (mRNA shownin SEQ ID NO: 21463; fully modified with 5-methylcytosine and1-methylpseudouridine; 5′ cap, cap 1, polyA tail of approximately 160nucleotides (not shown in sequence)) or a control of G-CSF mRNA (mRNAshown in SEQ ID NO: 21438; fully modified with 5-methylcytosine andpseudouridine; 5′ cap, cap1; polyA tail of approximately 160 nucleotidesnot shown in sequence) are added directly after seeding the cells atquantities of 4000, 800, 400, 40, and 4 ng per well and incubated. G-CSFmRNA transfected cells were treated with anti-LDLR antibody and one setof LDLR transfected cells were treated with normal goat IgG as controls.Bound primary antibodies were detected by FACS analysis followingtreatment with a PE-labeled secondary antibody.

As shown in FIG. 66A, results of the FACS analyses shows 74.8% of allgated live cells were detected to express LDLR at the 800 ng dose ofLDLR mRNA. At the 40 ng dose of LDLR mRNA, 11.6% of all gated live cellswere detected to express LDLR. No staining was observed in LDLR mRNAtreated cells stained with the control nonimmune IgG. No LDLR positivecells were detected in cells transfected with G-CSF mRNA.

B. Protein Accumulation

Human embryonic kidney epithelial (HEK293) cells (LGC standards GmbH,Wesel, Germany) are seeded on 6-well plates (BD Biosciences, San Jose,USA). HEK293 are seeded at a density of about 500,000 cells per well in3 mL cell culture medium. Formulations containing LDLR mRNA (mRNA shownin SEQ ID NO: 21463; fully modified with 5-methylcytosine and1-methylpseudouridine; 5′ cap, cap 1, polyA tail of approximately 160nucleotides (not shown in sequence)) or a control of G-CSF mRNA (mRNAshown in SEQ ID NO: 21438; fully modified with 5-methylcytosine andpseudouridine; 5′ cap, cap1; polyA tail of approximately 160 nucleotidesnot shown in sequence) are added directly after seeding the cells perwell and incubated. Fifteen hours later the transfection media isreplaced with complete media. Transfected cells are harvested at 0, 2,4, 8, 24, 48, and 72 hours after media replacement. Transfected cellswere treated with anti-LDLR antibody conjugated to Phycoerythrin (PE)and one set of LDLR transfected cells were treated with normal goat IgGconjugated to PE as controls. Bound primary antibodies were detected byFACS analysis as described above.

As shown in FIG. 66B, the FACS analysis shows ˜65% of all gated livecells were detected to express LDLR at the 0.0-h time point (15.0-hafter transfection) after washing away the transfection media. Thepercent positive cells declined with time at 37° C., such that by 24hpost-removal of the transfection media, LDLR was not detected.

C. BODIPY®-Labeled LDLR

To evaluate whether the expressed LDLR were functional, BODIPY®-labeledLDL was used. HEK293 cells were transfected overnight with either LDLRmodified mRNA (mRNA shown in SEQ ID NO: 21463; fully modified with5-methylcytosine and 1-methylpseudouridine; 5′ cap, cap 1, polyA tail ofapproximately 16 nucleotides (not shown in sequence)) or G-CSF modifiedmRNA (mRNA shown in SEQ ID NO: 21438; fully modified with5-methylcytosine and pseudouridine; 5′ cap, cap1; polyA tail ofapproximately 160 nucleotides not shown in sequence), the cells werewashed and incubated with increasing amounts of BODIPY-LDL. Followingincubation for 1.0-h at 37° C., the cells were washed and the binding ofBODIPY-LDL was assessed by FACS. Binding of BODIPY-LDL to LDLR mRNAtranfected cells was high affinity (Kd ˜60 ng/mL) and saturable as shownin FIG. 66C.

In competition studies, binding of BODIPY-LDL could be reduced in adose-dependent manner by unlabeled LDL (FIG. 66D). These data show thatbinding of BODIPY-LDL to cells expressing LDLR mRNA is saturable,specific, and of high affinity.

Example 191 UDP glucuronosyltransferase 1 family, polypeptide Al(UGT1A1) Expression

A. In Vitro UGT1A1 Expression

Human embryonic kidney epithelial (HEK293) cells (LGC standards GmbH,Wesel, Germany) are seeded on 6-well plates (BD Biosciences, San Jose,USA). HEK293 are seeded at a density of about 500,000 cells per well in3 mL cell culture medium. Formulations containing UDPglucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) mRNA (mRNAshown in SEQ ID NO: 21463; fully modified with 5-methylcytosine and1-methylpseudouridine; 5′ cap, cap 1, polyA tail of approximately 160nucleotides (not shown in sequence) or a control of G-CSF mRNA (mRNAshown in SEQ ID NO: 21438; fully modified with 5-methylcytosine andpseudouridine; 5′ cap, cap1; polyA tail of approximately 160 nucleotidesnot shown in sequence) are added directly after seeding the cells atquantities of 4000, 800, 400, 40, and 4 ng per well and incubated. G-CSFmRNA transfected cells were treated with anti-UGT1A1 antibody and oneset of UGT1A1 transfected cells were treated with normal goat IgG ascontrols. Bound primary antibodies were detected by FACS analysisfollowing treatment with a PE-labeled secondary antibody.

As is shown in FIG. 67 the percent positive cells measured by FACSincreased with the amount of UGT1A1 mRNA transfected into the HEK293cells and reached a maximum at 74.8% of all gated live cells at the 800ng dose of UGT1A1 mRNA. No further increase was in percent positivecells was observed at the highest dose tested (4000 ng). These datademonstrate that the mRNA delivery and/or expression in mammalian cellsare saturable. No staining was observed in UGT1A1 mRNA treated cellsstained with the control nonimmune IgG and no UGT1A1 positive cells weredetected in cells transfected with G-CSF mRNA.

B. In Vitro UGT1A1 Expression—Protein Accumulation

Human embryonic kidney epithelial (HEK293) cells (LGC standards GmbH,Wesel, Germany) are seeded on 6-well plates (BD Biosciences, San Jose,USA). HEK293 are seeded at a density of about 500,000 cells per well in3 mL cell culture medium. Formulations containing UGT1A1 mRNA (mRNAshown in SEQ ID NO: 2146; fully modified with 5-methylcytosine and1-methylpseudouridine; 5′ cap, cap 1, polyA tail of approximately 160nucleotides (not shown in sequence) or a control of G-CSF mRNA (mRNAshown in SEQ ID NO: 21438; fully modified with 5-methylcytosine andpseudouridine; 5′ cap, cap1; polyA tail of approximately 160 nucleotidesnot shown in sequence) are added directly after seeding the cells perwell and incubated. Fifteen hours later the transfection media isreplaced with complete media. Transfected cells are harvested at 0, 2,4, 8, 24, 48, and 72 hours after media replacement. Transfected cellswere treated with anti-UGT1A1 antibody conjugated to Phycoerythrin (PE)and one set of UGT1A1 transfected cells were treated with normal goatIgG conjugated to PE as controls. Bound primary antibodies were detectedby FACS analysis as described above.

As is shown in FIG. 68, UGT1A1 protein accumulation in the UGT1A1 mRNAtransfected cells increased with time of incubation after mediareplacement to a maximum at 24 hours and then declined to baselinevalues by 72 hours. Under these conditions, the apparent half-life ofUGT1A1 mRNA was approximately 40.0-h.

Example 192 Detection of UGT1A1 and OTC by Western blot

HEK293 Human embryonic kidney epithelial (HEK293) cells (LGC standardsGmbH, Wesel, Germany) are seeded on 6-well plates (BD Biosciences, SanJose, USA). HEK293 are seeded at a density of about 500,000 cells perwell in 2.4 mL cell culture medium. Formulations containing UGT1A1 mRNA(mRNA shown in SEQ ID NO: 21464; fully modified with 5-methylcytosineand 1-methylpseudouridine; 5′ cap, cap 1, polyA tail of approximately160 nucleotides (not shown in sequence) or OTC mRNA (mRNA shown in SEQID NO: 1659; fully modified with 5-methylcytosine and1-methylpseudouridine; 5′ cap, cap1; polyA tail of approximately 160nucleotides not shown in sequence) are added directly after seeding thecells at quantities of 800 ng per well and incubated for 16 hours. Cellswere washed with 500 μL PBS and harvested through cell scraping into 500μL PBS. Cells were centrifuged at 200×g for 5 minutes and resuspended in250 μL RIPA buffer supplemented with protease inhibitors. The tubes werecentrifuged for ten minutes at 14,000×g. The supernatants weretransferred to centrificon concentration columns and centrifuged for 30minutes at 14,000×g to concentrate the lysate. Lysate was quantifiedthrough BCA protein assay (Thermo Fisher Scientific Inc, Rockford, USA).

Protein lysates were loaded on NuPage SDS-PAGE system (chambers andpower supply) with 1.5 mm ready-to-use Bis-Tris gels and 4-12%acrylamide gradient with MOPS-buffer as running aid (all from LifeTechnologies, Grand Island, N.Y.). Each lysate sample was prepared to 40ul final volume. This sample contained 25 ug protein lysate in variablevolume, RIPA buffer to make up volume to 26 μl, 4 μl of 10× reducingagent and 10 ul 4× SDS loading buffer (both from Life Technologies,Grand Island, N.Y.). Samples were heated at 95° C. for 5 minutes andloaded on the gel. Standard settings were chosen by the manufacturer,200V, 120 mA and maximum 25W. Run time was 60 minutes, but no longerthan running dye reaching the lower end of the gel.

After the run was terminated, the plastic case was cracked and theencased gel transferred to a ready-to-use nitrocellulose membrane kitand power supply (iBLOT; LifeTechnologies, Grand Island, N.Y.). Usingdefault settings, the protein lysate was transferred by high Ampereelectricity from the gel to the membrane.

After the transfer, the membranes were incubated in 5% bovine serumalbumin (BSA) in 1× tris buffered saline (TBS) for 15 minutes then in 5%BSA in 1×TBS+0.1% Tween for another 15 minutes. Primary antibodies, goatanti-human UGT1A1 (R&D systems) and rabbit anti-human OTC(NBP1) wereapplied in 3 ml of 5% BSA in 1× TBS solution at a 1:500 dilution for 3hours at room temperature and gentle agitation on an orbital shaker. TheOTC primary antibodies were detected with the WesternBreeze ChromogenicKit—Anti-Rabbit according to the manufacturing protocol, and the UGT1A1primary antibodies were detected with the WesternBreeze ChromogenicKit—Anti-Goat (Invitrogen, Grand Island, N.Y.) according to themanufacturing protocol.

As is shown in FIG. 69, UGT1A1 was detected as a ˜60 kDa band byanti-UGT1A1 IgG in lysates of cells transfected with UGT1A1 mRNA (lane4) but not in cells transfected with OTC mRNA (lane 3). Using the samelysates, OTC was detected as a ˜39 kDa band by anti-OTC IgG in lysatesof cells transfected with OTC mRNA (lane 7) but not in cells transfectedwith UGT1A1 mRNA (lane 8). These data establish that exogenous UGT1A1and OTC mRNAs can direct the synthesis of their cognate proteins inmammalian cells.

Example 193 PAH and UGT1A1Expression and Detection

A. HEK293, Mouse and Rat Myoblast Cell Transfection with

HEK293 (LGC standards GmbH, Wesel, Germany), C2C12 mouse myoblast (ATCC)and L6 rat myoblast (ATCC) cell lines were plated into 24-well dishes(BD Biosciences, San Jose, USA) (1.5×10⁵ cells/well)) in DMEM (ATCC)supplemented with 10% fetal calf serum (ATCC).

Transfection solutions were prepared for each well to be treated bycombining 250 ng of PAH modified mRNA (SEQ ID NO: 1660; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1) fullymodified with 5-methylcytosine and pseudouridine, UGT1A1 modified mRNA(SEQ ID NO: 21464; polyA tail of approximately 160 nucleotides not shownin sequence; 5′ cap, Cap1; fully modified with 5-methylcytosine andpseudouridine or G-CSF modified mRNA (SEQ ID NO: 21438; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′ cap, Cap1; fullymodified with 5-methylcytosine and pseudouridine with 50 μl Opti-MEMreagent (Life Technologies, Grand Island, N.Y.) in a first tube and 1 μlof L2000 transfection reagent (Life Technologies, Grand Island, N.Y.) in50 μl of Opti-MEM in a second tube. After preparation, first and secondtubes were incubated at room temperature for 5 minutes before combiningthe contents of each. Combined transfection solutions were incubated for15 minutes at room temperature. 100 μl of transfection solution was thenadded to each well. Cells were cultured for an additional 14 hoursbefore continued analysis.

B. PAH, UGT1A1 Detection by Flow Cytometry

After transfection, medium were removed from cells and 60 μl of 0.25%trypsin (Life Technologies, Grand Island, N.Y.) was added to each well.Cells were trypsinized for 2 minutes before the addition of 240 μl/wellof trypsin inhibitor (Life Technologies, Grand Island, N.Y.). Resultingcell solutions were transferred to 96 well plates (Corning LifeSciences, Tewksbury, Mass.), cells were pelleted by centrifugation (800×gravity for 5 minutes) and supernatants were discarded. Cell pelletswere washed with PBS and resuspended in Foxp3 Fixation/Permeabilizationsolution (eBioscience, San Diego, Calif.) for 45 minutes. Cells werepelleted again by centrifugation (800× gravity for 5 minutes) andresuspended in permeabilization buffer (eBiosciences, San Diego, Calif.)for 10 minutes. Cells were pelleted again by centrifugation (800×gravity for 5 minutes) and washed in permeabilization buffer. PAHtransfected cells were treated with anti-PAH antibody conjugated toPhycoerythrin (PE). One set of PAH transfected cells were treated withnormal goat IgG conjugated to PE as staining controls. One set of G-CSFtransfected cells was treated with anti-PAH antibody conjugated toPhycoerythrin (PE) as a transfection control. Bound primary antibodieswere detected by FACS analysis as described above. Labeled cells werethen combined with FACS buffer (PBS with 1% bovine serum albumin and0.1% sodium azide) and transferred to cluster tubes and labeled cellswere then analyzed by flow cytometry using a BD Accuri (BD Biosciences,San Jose, Calif.). As is shown in FIG. 70, 11.9% of HEK293 cellstransfected with PAH were detected to express PAH, and 58.6% of HEK293cells transfected with UGT1A1 were detected to express UGT1A1. For eachmRNA, the percent positive cells were similar to the results obtainedwith HEK293 cells when rat or mouse myoblast cells were used.

Example 194 Expression of UGT1A1 in HEK293 Cells Using Direct CaptureCapillary Electrophoresis

HEK293 cells were transfected with UGT1A1 mRNA and cell lysates wereprepared as described above. Western analysis was performed using theSimple Simon Western system (Protein Simple). As is shown in FIG. 71,UGT1A1 (˜60 kDa band) was detected in lysates of cells transfected withUGT1A1 mRNA by the anti-UGT1A1 IgG (lanes 1, 4, and 6) but not by anidentical concentration of nonimmune IgG (lanes 2 and 5). Similarly, noUGT1A1 was detected in using an identical amount of cell lysate ofnon-transfected Hep3b cells (lane 3) or in lysates from HEK293 cellstransfected with OTC mRNA (lane 7).

Normal C57BL6 mice were given a single intravenous injection of LNPcontaining UGT1A1 mRNA at a dose of 0.5 mg/kg. Twenty four hours laterthe mice were sacrificed and the livers were isolated from each mouse.Microsomal membrane extracts were prepared from each liver byhomogenization in ice-cold 5.0 mL of phosphate buffered saline (pH 7.4)containing protease inhibitors, using a Polytron homogenizer. The tissuehomogenate was mixed with 40 ml of ice-cold microsome buffer (2.62 mMKH2PO4, 1.38 mM K2HPO4, 2% glycerol, and 0.5 mM dithiothreitol) andcentrifuged at 12,000×g for 20 min at 4° C. The supernatant fraction wasthen centrifuged at 100,000×g for 60 min at 4° C. The microsomal pelletwas resuspended in microsome buffer and the protein concentration wasdetermined by the Bradford method. Microsomal extracts werecharacterized by detection of a calnexin band by Western blotting usinganti-calnexin antibody (Assay Designs, Ann Arbor, Mich.). Approximately2-5 μg of microsomal extract was applied to the capillaries andelectrophoresis and protein immobilization was performed as described bythe manufacturer. As is shown in FIG. 72 UGT1A1 was detected inmicrosomal extracts of mice treated with LNPs containing UGT1A1 mRNAusing an antibody specific (described above) for UGT1A1(lane 2) but notwith a non-specific antibody (lane 4). The molecular weight of theUGT1A1 in mouse liver was the same as that observed with extracts ofHEK293 cells transfected with UGT1A1mRNA and probed with the anti-UGT1A1IgG (lane 3). No UGT1A1 band was detected in extracts of HEK293 cellstransfected with OTC mRNA (lane 5). These data demonstrate expression ofUGT1A1 mRNA in vivo in mice injected with LNPs containing UGT1A1 mRNA.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

1. A method of producing a polypeptide of interest in a mammalian cellor tissue comprising contacting said mammalian cell or tissue with amodified mRNA, wherein said modified mRNA encodes a member selected fromthe group consisting of glucosidase, beta, acid (SEQ ID NO: 1016),glucosidase, beta, acid (SEQ ID NO: 1017), glucosidase, beta, acid (SEQID NO: 1018), glucosidase, beta, acid (SEQ ID NO: 1019), glucosidase,beta, acid (SEQ ID NO: 1020), glucosidase, beta, acid (SEQ ID NO: 1021)and glucosidase, beta, acid (SEQ ID NO: 1022), and wherein said modifiedmRNA comprises modifications selected from the group consisting of: (a)5-methylcytosine and 2-thiouridine, (b) pseudouridine and5-methylcytosine, and (c) 1-methylpseudouridine.
 2. The method of claim1, wherein the modified mRNA comprises the modification1-methylpseudouridine and further comprises the modification5-methylcytosine.
 3. The method of claim 1, wherein the modified mRNAcomprises the modifications 5-methylcytosine and 2-thiouridine, wherein25% of the cytosine residues in the modified mRNA are 5-methylcytosinesand 25% of the uridine residues in the modified mRNA are 2-thiouridine.4. The method of claim 1, wherein at least one of the 5′ untranslatedregion or the 3′ untranslated region of the modified mRNA is not derivedfrom beta-globin.
 5. A modified mRNA encoding a polypeptide of interest,said polypeptide of interest selected from the group consisting ofglucosidase, beta, acid (SEQ ID NO: 1016), glucosidase, beta, acid (SEQID NO: 1017), glucosidase, beta, acid (SEQ ID NO: 1018), glucosidase,beta, acid (SEQ ID NO: 1019), glucosidase, beta, acid (SEQ ID NO: 1020),glucosidase, beta, acid (SEQ ID NO: 1021) and glucosidase, beta, acid(SEQ ID NO: 1022), and wherein said modified mRNA comprisesmodifications selected from the group consisting of: (a)5-methylcytosine and 2-thiouridine, (b) pseudouridine and5-methylcytosine, and (c) 1-methylpseudouridine.
 6. The modified mRNA ofclaim 5, wherein the modified mRNA comprises the modification1-methylpseudouridine and further comprises the modification5-methylcytosine.
 7. The modified mRNA of claim 5, wherein the modifiedmRNA comprises the modifications 5-methylcytosine and 2-thiouridine,wherein 25% of the cytosine residues in the modified mRNA are5-methylcytosines and 25% of the uridine residues in the modified mRNAare 2-thiouridine.
 8. The modified mRNA of claim 5, wherein at least oneof the 5′ untranslated region or the 3′ untranslated region of themodified mRNA is not derived from beta-globin.
 9. (canceled)